Lipid-encapsulated gas microsphere compositions and related methods

ABSTRACT

The invention provides, inter alia, improved lipid formulations used to generate lipid-encapsulated gas microspheres, and methods of their use.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/203,725 filed Jul. 6, 2016, pending, which is a continuation of andclaims the benefit under 35 U.S.C. § 120 and § 365(c) of InternationalApplication No. PCT/US2015/067615, with an international filing date ofDec. 28, 2015, which claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application Ser. No. 62/098,453, filed Dec. 31, 2014, eachof which is incorporated by reference herein in its entirety.

SUMMARY

The invention provides, in part, new and improved formulations formaking ultrasound contrast agents as well as preparations of ultrasoundcontrast agents themselves. Such formulations are less complex in theircomposition, their method of manufacture and their method of use and,surprisingly, more robust than prior art formulations used to makeultrasound contrast agents, including more stable at room temperaturefor extended periods of time. Such formulations can be used to makeultrasound contrast agents, surprisingly, without complex manipulation.

Provided herein are these new formulations, kits comprising these newformulations, methods of using these formulations including methods ofusing these formulations to make ultrasound contrast agents, andcompositions or preparations of the lipid-encapsulated gas microspheresthemselves. These new formulations include the non-aqueous mixturesdescribed in greater detail herein.

In one aspect, provided herein is a composition consisting of orconsisting essentially of a non-aqueous mixture of DPPA, DPPC andPEG5000-DPPE in propylene glycol and glycerol and a buffer.

In another aspect, provided herein is a composition consisting of orconsisting essentially of a non-aqueous mixture of DPPA, DPPC andPEG5000-DPPE in propylene glycol and a buffer.

In another aspect, provided herein is a composition consisting of orconsisting essentially of a non-aqueous mixture of DPPA, DPPC andPEG5000-DPPE in glycerol and a buffer.

The buffer may be, without limitation, an acetate buffer (e.g., acombination of sodium acetate and acetic acid), or a benzoate buffer(e.g., a combination of sodium benzoate and benzoic acid), or asalicylate buffer (e.g., a combination of sodium salicylate andsalicylic acid).

The foregoing compositions may be provided in a sterile container,optionally with a perfluorocarbon gas, and further optionally withinstructions for use including instructions for activating suchcompositions in the presence of a perfluorocarbon gas and optionally inthe presence of an aqueous diluent in order to generatelipid-encapsulated gas microspheres. The composition to be activated maycomprise the aqueous diluent as a second phase and thus may benon-homogeneous prior to activation.

In another aspect, provided herein is a composition comprising anon-aqueous mixture of DPPA, DPPC and PEG5000-DPPE in propylene glycoland glycerol, and a perfluorocarbon gas.

In some embodiments, the weight to weight to weight (w/w/w) ratio ofDPPA, DPPC and PEG5000-DPPE (combined) to propylene glycol to glycerolis in a range of about 1:50:50 to about 1:1000:1000, or about 1:100:100to about 1:600:700. In some embodiments, the w/w/w ratio of DPPA, DPPCand PEG5000-DPPE (combined) to propylene glycol to glycerol is about1:120:120 to about 1:400:400, or about 1:120:120 to about 1:300:300, orabout 1:120:120 to about 1:250:250. In some embodiments, the w/w/w ratioof DPPA, DPPC and PEG5000-DPPE (combined) to propylene glycol toglycerol is about 1:100:150 to about 1:150:200. In some embodiments, thew/w/w ratio of DPPA, DPPC and PEG5000-DPPE (combined) to propyleneglycol to glycerol is about 1:250:300 to about 1:300:350. In someembodiments, the w/w/w ratio of DPPA, DPPC and PEG5000-DPPE (combined)to propylene glycol to glycerol is about 1:500:600 to about 1:600:700.In some embodiments, the w/w/w ratio of DPPA, DPPC and PEG5000-DPPE(combined) to propylene glycol to glycerol is about 1:138:168 or about1:276:336 or about 1:552:673.

In some embodiments, the w/w/w ratio of DPPA, DPPC and PEG5000-DPPE(combined) to propylene glycol to glycerol is about 0.75 mg: 103.5 mg:126.2 mg. In some embodiments, the w/w/w ratio of DPPA, DPPC andPEG5000-DPPE (combined) to propylene glycol to glycerol is about 0.375mg: 103.5 mg: 126.2 mg. In some embodiments, the w/w/w ratio of DPPA,DPPC and PEG5000-DPPE (combined) to propylene glycol to glycerol isabout 0.1875 mg: 103.5 mg: 126.2 mg.

In another aspect, provided herein is a composition comprising anon-aqueous mixture of DPPA, DPPC and PEG5000-DPPE in propylene glycol,and a perfluorocarbon gas.

In some embodiments, the w/w ratio of DPPA, DPPC and PEG5000-DPPE(combined) to propylene glycol is in a range of about 1:10 to about1:2000, or about 1:10 to about 1:1500, or about 1:10 to about 1:1000, orabout 1:20 to about 1:2000, or about 1:50 to about 1:1000, or about 1:50to about 1:600, or about 1:100 to about 1:600.

In some embodiments, the w/w ratio of DPPA, DPPC and PEG5000-DPPE(combined) to propylene glycol is about 1:100 to about 1:200, or about1:100 to about 1:150. In some embodiments, the w/w ratio of DPPA, DPPCand PEG5000-DPPE (combined) to propylene glycol is about 1:200 to about1:350, or about 1:250 to about 1:300. In some embodiments, the w/w ratioof DPPA, DPPC and PEG5000-DPPE (combined) to propylene glycol is about1:500 to about 1:600, or about 1:525 to about 1:575. In someembodiments, the w/w ratio of DPPA, DPPC and PEG5000-DPPE (combined) topropylene glycol is about 1:138 or about 1:276 or about 1:552.

In some embodiments, the w/w ratio of DPPA, DPPC and PEG5000-DPPE(combined) to propylene glycol is about 0.75 mg: 103.5 mg or about 0.375mg: 103.5 mg or about 0.1875 mg: 103.5 mg.

In another aspect, provided herein is a composition comprising anon-aqueous mixture of DPPA, DPPC and PEG5000-DPPE in glycerol, and aperfluorocarbon gas.

In some embodiments, the w/w ratio of DPPA, DPPC and PEG5000-DPPE(combined) to glycerol is in a range of about 1:10 to about 1:2000, orabout 1:15 to about 1:1500, or about 1:50 to about 1:1000, or about 1:50to about 1:7000, or about 1:100 to about 1:700. In some embodiments, thew/w ratio of DPPA, DPPC and PEG5000-DPPE (combined) to glycerol is about1:100 to about 1:200 or about 1:125 to about 1:175. In some embodiments,the w/w ratio of DPPA, DPPC and PEG5000-DPPE (combined) to glycerol isabout 1:250 to about 1:400, or about 1:300 to about 1:350. In someembodiments, the w/w ratio of DPPA, DPPC and PEG5000-DPPE (combined) toglycerol is about 1:550 to about 1:700 or about 1:650 to about 1:700. Insome embodiments, the w/w ratio of DPPA, DPPC and PEG5000-DPPE(combined) to glycerol is about 1:168 or about 1:336 or about 1:673.

In some embodiments, the w/w ratio of DPPA, DPPC and PEG5000-DPPE(combined) to glycerol is about 0.75 mg: 126.2 mg, or about 0.375 mg:126.2 mg, or about 0.1875 mg: 126.2 mg.

In other aspects, provided herein is a container comprising any of theforegoing compositions.

In some embodiments, the container is a single chamber container.

In some embodiments, the container comprises a first and a secondchamber, and wherein the non-aqueous mixture is in the first chamber andthe perfluorocarbon gas is in the second chamber.

In other aspects, provided herein is a container comprising any of theforegoing compositions in a first chamber and an aqueous diluent in asecond chamber.

In other aspects, provided herein is a container comprising any of theforegoing compositions and an aqueous diluent, wherein the non-aqueousmixture is provided in a first chamber, the perfluorocarbon gas isprovided in a second chamber, and the aqueous diluent is provided in athird chamber.

In some embodiments, the aqueous diluent is an aqueous saline solution.In some embodiments, the aqueous diluent is an aqueous bufferedsolution. In some embodiments, the aqueous diluent is an aqueousbuffered saline solution.

In another aspect, provided herein is a composition comprising a mixtureof DPPA, DPPC and PEG5000-DPPE in solid form, and a perfluorocarbon gas.The mixture of DPPA, DPPC and PEG5000-DPPE in solid form may be ablended solid form (e.g., a relatively homogeneous mixture of thelipids) or it may be a combination of the solid forms of each lipid(e.g., which may or may not be a homogeneous mixture of the lipids). Inanother aspect, provided herein is a container comprising the foregoingsolid form composition. In some embodiments, the container is acontainer having a single chamber. In some embodiments, the container isa container having two chambers, wherein a first chamber comprises DPPA,DPPC and PEG5000-DPPE in solid form, and a second chamber comprises theperfluorocarbon gas. In some embodiments, the container is a containerhaving two chambers, wherein a first chamber comprises DPPA, DPPC andPEG5000-DPPE in solid form and the perfluorocarbon gas, and a secondchamber comprises (a) propylene glycol, (b) propylene glycol andglycerol, or (c) glycerol. The w/w/w ratios of the lipids combined topropylene glycol and/or to glycerol may be as stated above. In someembodiments, the container is a container having three chambers, whereina first chamber comprises DPPA, DPPC and PEG5000-DPPE in solid form, asecond chamber comprises the perfluorocarbon gas, and a third chambercomprises (a) propylene glycol, (b) propylene glycol and glycerol, or(c) glycerol. In some embodiments, the container is a container havingan additional chamber comprising an aqueous diluent.

In another aspect, provided herein is a composition comprisinglipid-encapsulated gas microspheres comprising DPPA, DPPC andPEG5000-DPPE and perfluorocarbon gas, in a non-aqueous solutioncomprising propylene glycol and glycerol.

In another aspect, provided herein is a composition comprisinglipid-encapsulated gas microspheres comprising DPPA, DPPC andPEG5000-DPPE, in a non-aqueous solution comprising propylene glycol.

In another aspect, provided herein is a composition comprisinglipid-encapsulated gas microspheres comprising DPPA, DPPC andPEG5000-DPPE and perfluorocarbon gas, in a non-aqueous solutioncomprising glycerol.

In some embodiments, the lipid-encapsulated gas microspheres have anaverage diameter ranging from about 1.0 microns to about 2.0 microns. Insome embodiments, the lipid-encapsulated gas microspheres have anaverage diameter ranging from about 1.2 microns to about 2.0 microns. Insome embodiments, the lipid-encapsulated gas microspheres have anaverage diameter of about 1.4 to 1.8 microns.

In some embodiments, the lipid-encapsulated gas microspheres are presentin the composition at a concentration of greater than 10⁸/mL.

Various embodiments apply equally to the foregoing compositions and willbe recited now.

In some embodiments, the non-aqueous mixture comprises less than 5% ofwater by weight (i.e., weight of water to weight of the combination oflipid and propylene glycol and/or glycerol). In some embodiments, thenon-aqueous mixture comprises 1-4% water by weight. In some embodiments,the non-aqueous mixture comprises less than 1% water by weight.

In some embodiments, the composition is salt-free, meaning that it maycomprise the counter-ions to the lipids in the composition but is freeof other ions. The lipid counter-ions are typically cations such assodium. Thus, in some embodiments the composition does not compriseanions. In some embodiments, the composition is free of sodium chloride.In some embodiments, the composition is free of chloride ions.

In some embodiments, the composition further comprises a buffer. In someembodiments, the composition further comprises a non-phosphate buffer.In some embodiments, the composition further comprises an acetatebuffer, or a benzoate buffer, or a salicylate buffer.

In some embodiments, DPPA, DPPC and PEG5000-DPPE combined are present ina concentration of about 0.9 to about 8 mg lipid per ml of non-aqueousmixture, about 0.9 mg to about 7.5 mg lipid per ml non-aqueous mixture,about 2 mg to about 7.5 mg lipid per ml non-aqueous mixture, or about 2mg to about 4 mg lipid per ml non-aqueous mixture. In some embodiments,DPPA, DPPC and PEG5000-DPPE combined are present in a concentration ofabout 0.94 mg to about 7.5 mg lipid per ml of non-aqueous mixture, orabout 1.875 mg to about 7.5 mg lipid per ml of non-aqueous mixture,including about 1.875 mg to about 3.75 mg lipid per ml of non-aqueousmixture, and about 3.75 to about 7.5 mg lipid per ml of non-aqueousmixture. In some embodiments, DPPA. DPPC and PEG5000-DPPE are present ina ratio of about 10:82:8 (mole %).

In some embodiments, the non-aqueous mixture, alone or in combinationwith a perfluorocarbon gas, comprises less than 5% impurities whenstored at room temperature for about 3 months. In some embodiments, thenon-aqueous mixture, alone or in combination with a perfluorocarbon gas,comprises fewer impurities than DEFINITY® when both are stored at roomtemperature (i.e., when the composition and DEFINITY® are stored at roomtemperature).

In some embodiments, the perfluorocarbon gas is perfluoropropane gas.

In some embodiments, PEG5000-DPPE is MPEG5000-DPPE.

In some embodiments, the composition is provided in a vial. In someembodiments, the composition is provided in a vial with an actual volumeof less than or equal to about 3.8 ml.

In some embodiments, the composition is provided in a vial with aV-bottom. In some embodiments, the composition is provided in a vialwith a flat-bottom. In some embodiments, the composition is provided ina vial with a rounded-bottom. In some embodiments, the vial is a glassvial. In some embodiments, a composition comprising a non-aqueousmixture of DPPA, DPPC and PEG5000-DPPE combined in propylene glycol andglycerol, and a perfluorocarbon gas, is provided in a 2 ml Nipro(Wheaton) vial at a lipid concentration of about 3.75 mg/ml. In someembodiments, a composition comprising a non-aqueous mixture of DPPA,DPPC and PEG5000-DPPE combined in propylene glycol and glycerol, and aperfluorocarbon gas, is provided in a 2 ml Schott vial at a lipidconcentration of about 3.75 mg/ml.

In some embodiments, the composition is provided in a single chambercontainer. In some embodiments, the composition is provided in amultiple chamber container. In some embodiments, the composition isprovided in a first chamber and an aqueous diluent is provided in asecond chamber. The aqueous diluent may be a saline solution or it maybe saline-free. The aqueous diluent may be buffered solution or it maybe buffer-free. The aqueous diluent may be a buffered saline solution.

In another aspect, provided herein is a kit comprising any of theforegoing compositions in a container. In some embodiments, thecontainer is a single chamber container.

In some embodiments, the kit comprises a second container. In someembodiments, the second container comprises an aqueous diluent. In someembodiments, the second container is a pre-filled syringe.

In some embodiments, the container is a multi-chamber container. In someembodiments, the first container comprises the lipids (i.e., DPPA, DPPCand PEG5000-DPPE) in solid form, and the second container comprisespropylene glycol or glycerol or propylene glycol and glycerol. A thirdcontainer may comprise an aqueous diluent.

In some embodiments, the first container comprises the lipids inpropylene glycol, and the second container comprises glycerol or aqueousdiluent. Alternatively, the second container comprises glycerol and athird container comprises aqueous diluent.

In some embodiments, the first container comprises the lipids inglycerol, and the second container comprises propylene glycol or aqueousdiluent. Alternatively, the second container comprises propylene glycoland a third container comprises aqueous diluent.

In some embodiments, the first container comprises the lipids inpropylene glycol and glycerol, and the second container comprisesaqueous diluent.

In some embodiments, the kit further comprises an activation device suchas but not limited to a VIALMIX® device.

It also has been found according to the invention that certain of thenon-aqueous mixtures (i.e., certain of these modified lipidformulations) may be used to generate lipid-encapsulated gasmicrospheres, through a process referred to herein as “activation”,either as a non-aqueous mixture or following simple addition of aqueousdiluent without regard to the degree of homogeneity of the combinedsolution. This was surprising because certain marketed contrast agentsare made by activating a pre-formulated, single-phase mixture comprisinglipids in excess aqueous solution. It was not known prior to theinvention that lipid-encapsulated microspheres of suitable size andnumber could be generated without either pre-formulating the lipid in anaqueous solution or in the absence of aqueous solution.

Thus, in another aspect, provided herein is a method of forming anultrasound contrast agent comprising activating any of the foregoingnon-aqueous mixtures in the presence of a perfluorocarbon gas, and inthe presence or absence of aqueous diluent, to form lipid-encapsulatedgas microspheres.

In another aspect, provided herein is a method of forming an ultrasoundcontrast agent comprising combining any of the foregoing non-aqueousmixtures with an aqueous diluent in the presence of a perfluorocarbongas, and activating the combination to form lipid-encapsulated gasmicrospheres. The aqueous diluent may be added to the non-aqueousmixture with or without agitation or other modification (e.g., heating,etc.), and such combined mixture may be activated, in the presence of aperfluorocarbon gas, regardless of whether it is a single-phase mixture(i.e., the lipid and aqueous phases have been substantially commingledand/or the mixture appears relatively homogeneous) or a double-phasemixture (i.e., the lipid and aqueous phases have not been substantiallycommingled and/or the mixture does not appear relatively homogeneous).

In another aspect, provided herein is a method of forming an ultrasoundcontrast agent comprising combining certain of the foregoing non-aqueousmixtures with propylene glycol alone or propylene glycol and an aqueousdiluent (simultaneously or consecutively), and activating thecombination in the presence of perfluorocarbon gas to formlipid-encapsulated gas microspheres.

In another aspect, provided herein is a method of forming an ultrasoundcontrast agent comprising combining certain of the foregoing non-aqueousmixtures with glycerol alone or glycerol and an aqueous diluent(simultaneously or consecutively), and activating the combination in thepresence of perfluorocarbon gas to form lipid-encapsulated gasmicrospheres.

The non-aqueous mixtures may be at room temperature and/or may have beenstored at room temperature prior to use. Storage at room temperature mayhave ranged from days, to months, to years.

In some embodiments, activation occurs for 20-45 seconds. In someembodiments, activation occurs for 60-120 seconds.

In some embodiments, the method further comprises diluting thelipid-encapsulated gas microspheres in additional aqueous diluent.

In some embodiments, the method further comprises administering thelipid-encapsulated gas microspheres to a subject in need of contrastultrasound imaging.

In some embodiments, the composition is in a vial. In some embodiments,the composition is in a syringe. In some embodiments, the composition isin a single chamber container. In some embodiments, the composition isin a multi-chamber container.

In still other aspects, provided herein are methods for detecting and/ormeasuring levels of impurities in any one of the compositions describedherein. Such methods are particularly useful for assessing the integrityof a composition, and may be used to determine that the composition issuitable for use or should be discarded. The methods may be performed ona newly manufactured batch of the compositions described herein, or theymay be performed on a batch that has been in transit or in storage for aperiod of time following its manufacture.

The method comprises detecting and identifying components of the sample.In some embodiments, the method further comprises separating thecomponents based on physicochemical properties such as but not limitedto charge and lipophilicity, optionally prior to detection andidentification. In some embodiments, separation is performed prior todetection and the sample is diluted with saline prior to separation. Insome embodiments, the sample is mixed until a homogenous solution isobtained. Separation techniques based on physicochemical properties areknown in the art and include but are not limited to HPLC such as reversephase HPLC. Impurities are then detected and optionally measured usingtechniques such as but not limited to charged aerosol detection (CAD).In another embodiment, evaporative light scattering detection (ELSD) maybe used following separation. An example of such a detection isdescribed in greater detail herein.

These and other aspects and embodiments of the invention will bedescribed in greater detail herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Stability of a lipid blend/propylene glycol (LB/PG) formulationversus DEFINITY®.

FIG. 2. Stability of a lipid blend/propylene glycol/glycerol (LB/PG/G)formulation versus DEFINITY®.

FIG. 3. Stability of a lipid blend/propylene glycol/glycerol/buffer(LB/PG/G/buffer) formulation versus DEFINITY®.

DETAILED DESCRIPTION

It has been found, according to the invention, that lipid formulationsfor generating lipid-encapsulated gas microspheres to be used asultrasound imaging agents can be maintained at room temperature,including at room temperature for extended periods of time, withoutsignificant degradation. Previously, it was thought that lipidformulations to be used for the same purpose had to be stored at 4° C.in order to avoid degradation. It has been found, according to theinvention, that storage of these modified lipid formulations at roomtemperature for several months results in less than 5% impurities, alevel less than that present in a currently marketed ultrasound contrastagent when stored at room temperature for the same period of time.

Importantly, storage of these modified lipid formulations, referred toas lipid-containing non-aqueous mixtures, at room temperature, includinglong-term storage at room temperature, does not negatively impact theirability to form microspheres for use as ultrasound contrast agents, asevidenced by the ability to form microspheres of size and quantitycomparable to currently marketed ultrasound contrast agents. Thesemodified lipid formulations are therefore more robust than certainmarketed lipid formulations, at least in view of this enhancedstability.

The new lipid formulations described herein are easier to use thancertain existing formulations at least in part because they do notrequire refrigeration. In contrast, certain currently marketed lipidformulations must be refrigerated throughout their storage period, butthen are administered to patients at room temperature. This means thatsuch formulations must first be warmed from about 4° C. to about roomtemperature before they can be used. In contrast, the modified lipidformulations provided herein can be used essentially “off the shelf”without waiting a required period of time to warm to room temperature.This renders these modified formulations easier to use and alsofacilitates their immediate use in, for example, emergency situations.

In addition, due to the inherently more robust nature of the modifiedlipid formulations, there is less chance that their integrity has beencompromised prior to use, including for example during transport andstorage. In current practice, if certain of the marketed formulationshave been stored for any significant period of time at room temperature,then such formulations may be of questionable quality, and thus may needto be discarded. With the new formulations, an end user need not be asconcerned about the history or treatment of the formulation. Thus, apartfrom increased ease of use, there should also be less of the modifiedlipid formulations wasted due to integrity concerns.

These modified lipid formulations are intended for use as ultrasoundcontrast agents or as intermediates thereof. As such, and as describedherein, when provided together with a gas, they may be activated to formlipid-encapsulated gas microspheres with or without an aqueous diluent.Moreover, when an aqueous diluent is used, such formulations they may beactivated following simple addition of the aqueous diluent without anyneed for pre-formulation or pre-mixing of the non-aqueous mixture andthe aqueous diluent. As an example, addition of the aqueous diluent mayresult in a heterogeneous or two-phase mixture and this two-phasemixture may be activated. Certain marketed ultrasound contrast agentsare provided as pre-formulated, relatively homogeneous, single-phasemixtures of lipids formulated in an aqueous matrix, and are activated inthis essentially aqueous-formulated form. In contrast, the modifiedlipid formulations provided herein may be activated in their non-aqueousform or may be activated following simple addition of an aqueous diluentwith no requirement for pre-formulation of the lipid(s) and the aqueousdiluent or for the mixture to be homogeneous. This in turn means thatthe lipid formulation volume can be much smaller at the time ofactivation (and at the time of shipment and storage), and if necessaryit can be diluted just prior to use. This also means that theformulation integrity is less likely to be compromised because it ispossible to activate without adding an aqueous diluent, and then if theformulation is not used simply store the formulation for later use. Ifinstead the non-aqueous mixture had to be combined with an aqueoussolution in order to activate, then this type of flexibility would belost in this circumstance, and the formulation would have to bediscarded, again leading to unnecessary waste.

Accordingly, the invention is based, in part, on the unexpected andsurprising finding that lipids used to make lipid-encapsulated gasmicrospheres, that are themselves suitable as ultrasound contrastagents, when formulated in a non-aqueous mixture, can be stored forextended periods of time at room temperature without significantdegradation. The non-aqueous mixture may comprise propylene glycol, orglycerol, or a mixture of propylene glycol and glycerol. Importantly,the lipid formulations provided herein produce lipid-encapsulated gasmicrospheres on par with those produced by the currently marketedultrasound contrast agent, DEFINITY® (Perflutren Lipid MicrosphereInjectable Suspension), particularly with respect to microsphereconcentration and size, both of which impact the acoustic properties ofthe microspheres. Such lipid formulations are more robust andinsensitive to storage, including long term storage, at room temperaturethan DEFINITY®.

DEFINITY® is an ultrasound contrast agent that is approved by the FDAfor use in subjects with suboptimal echocardiograms to opacify the leftventricular chamber and to improve the delineation of the leftventricular endocardial border. DEFINITY® is provided in a vialcomprising a single phase solution comprising DPPA, DPPC andMPEG5000-DPPE in a 10:82:8 mole % ratio in an aqueous solution, and aheadspace comprising perfluoropropane gas. Prior to its administrationto a subject, DEFINITY® is activated by mechanical shaking (thereafterreferred to as “activated DEFINITY®”). Activation results in theformation of a sufficient number of lipid-encapsulated gas microsphereshaving an average diameter of 1.1 to 3.3 microns. DEFINITY® however mustbe refrigerated until just prior to use. This limits its utilityparticularly in settings that lack appropriate refrigeration,particularly during the storage period.

Provided herein are, inter alia, compositions for use in the manufactureof lipid-encapsulated gas microspheres and compositions and uses of thelipid-encapsulated gas microspheres themselves. The invention furtherprovides methods of manufacture of such microspheres.

Storage Formulations

These new formulations comprise a non-aqueous mixture of one or morelipids and propylene glycol (PG), or glycerol (G), or propylene glycoland glycerol (PG/G). It has been found, in accordance with theinvention, that these formulations may be stored at higher temperaturesfor longer periods of time than were previously possible using existingultrasound contrast agent formulations, without significant degradation.These compositions therefore may be used in a wider range of settingswithout particular concern about how the formulation was handled priorto use.

The enhanced stability of these new formulations is demonstrated in theExamples, where it is shown that lipid formulations in propylene glycolor propylene glycol and glycerol can be maintained for 3 months orlonger with less degradation than is observed in a DEFINITY® formulationmaintained at room temperature. The Examples demonstrate that theseformulations may be stored for about 3-6 months without significantdegradation.

The non-aqueous mixture of lipids in propylene glycol, or glycerol, orpropylene glycol and glycerol intends a mixture having less than orequal to 5% water by weight (i.e., weight of water to the weight of thecombination of lipids and propylene glycol and/or glycerol). In someinstances, the non-aqueous mixture comprises less than 5% water (w/w),1-4% water (w/w), 1-3% water (w/w), 2-3% water (w/w), or 1-2% water(w/w). In some instances, the non-aqueous mixture comprises less than 1%water (w/w). The water content may be measured at the end of manufacture(and prior to long term storage) or it may be measured after storage,including long term storage, and just before use.

The non-aqueous mixture also may be salt-free intending that it does notcontain any salts other than lipid counter-ions. More specifically, andas an example, lipids such as DPPA and DPPE are typically provided assodium salts. As used herein, a salt-free non-aqueous mixture maycomprise such counter-ions (e.g., sodium if DPPA and/or DPPE are used)but they do not contain other ions. In some instances, the non-aqueousmixture is free of sodium chloride or chloride.

The non-aqueous mixture may comprise a buffer. The buffer may be anacetate buffer, a benzoate buffer, or a salicylate buffer, although itis not so limited. Non-phosphate buffers are preferred in some instancesdue to their dissolution profiles in the non-aqueous mixtures providedherein. In some instances, a phosphate buffer may be used (e.g.,following or concurrent with addition of aqueous diluent).

In some embodiments, the non-aqueous mixture comprises, consists of, orconsists essentially of (a) one or more lipids, (b) propylene glycol, orglycerol, or propylene glycol/glycerol, and (c) a non-phosphate buffer.Such non-aqueous mixtures may be provided together with a gas such as aperfluorocarbon gas or they may be provided alone (i.e., in the absenceof a gas). Such non-aqueous mixtures may be provided in single useamounts and/or in single use containers, with or without a gas. Suchcontainers will typically be sterile.

The non-phosphate buffer may be, but is not limited to, an acetatebuffer, a benzoate buffer, a salicylate buffer, a diethanolamine buffer,a triethanolamine buffer, a borate buffer, a carbonate buffer, aglutamate buffer, a succinate buffer, a malate buffer, a tartratebuffer, a glutarate buffer, an aconite buffer, a citric buffer, anacetic buffer, a lactate buffer, a glycerate buffer, a gluconate buffer,and a tris buffer. In some instances, the buffer is a phosphate buffer.It is within the skill of the ordinary artisan to determine and optimizethe concentration of buffer for each buffer type.

Room temperature as used herein means a temperature of 15-30° C.,including 18-25° C. and 20-25° C., and all temperatures therebetween.The room temperature may be controlled (e.g., maintainedthermostatically) to be at such temperature but it is not so limited.

Lipids

These new formulations comprise one and typically more than one lipid.As used herein, “lipids” or “total lipid” or “combined lipids” means amixture of lipids.

The lipids may be provided in their individual solid state (e.g.,powdered) forms. Alternatively, the lipids may be provided as a lipidblend. Methods of making a lipid blend include those described in U.S.Pat. No. 8,084,056 and published PCT application WO 99/36104. A lipidblend, as used herein, is intended to represent two or more lipids whichhave been blended resulting in a more homogeneous lipid mixture thanmight otherwise be attainable by simple mixing of lipids in theirindividual powdered form. The lipid blend is generally in a powder form.A lipid blend may be made through an aqueous suspension-lyophilizationprocess or an organic solvent dissolution-precipitation process usingorganic solvents. In the aqueous suspension-lyophilization process, thedesired lipids are suspended in water at an elevated temperature andthen concentrated by lyophilization.

The organic solvent dissolution method involves the following steps:

(a) Contacting the desired lipids (e.g., DPPA, DPPC, and MPEG5000 DPPE)with a first non-aqueous solvent system. This system is typically acombination of solvents, for example CHCl₃/MeOH, CH₂Cl₂/MeOH, andtoluene/MeOH. Preferably, the first non-aqueous solvent is a mixture oftoluene and methanol. It may be desirable to warm the lipid solution toa temperature sufficient to achieve complete dissolution. Such atemperature is preferably about 25 to 75° C., more preferably about 35to 65° C. After dissolution, undissolved foreign matter may be removedby hot-filtration or cooling to room temperature and then filtering.Known methods of filtration may be used (e.g., gravity filtration,vacuum filtration, or pressure filtration).

(b) The solution is then concentrated to a thick gel/semisolid.Concentration is preferably done by vacuum distillation. Other methodsof concentrating the solution, such as rotary evaporation, may also beused. The temperature of this step is preferably about 20 to 60° C.,more preferably 30 to 50° C.

(c) The thick gel/semisolid is then dispersed in a second non-aqueoussolvent. The mixture is slurried, preferably near ambient temperature(e.g., 15-30° C.). Useful second non-aqueous solvents are those thatcause the lipids to precipitate from the filtered solution. The secondnon-aqueous solvent is preferably methyl t-butyl ether (MTBE). Otherethers and alcohols may be used.

(d) The solids produced upon addition of the second non-aqueous solventare then collected. Preferably the collected solids are washed withanother portion of the second non-aqueous solvent (e.g., MTBE).Collection may be performed via vacuum filtration or centrifugation,preferably at ambient temperature. After collection, it is preferredthat the solids are dried in vacuo at a temperature of about 20-60° C.

The contents of U.S. Pat. No. 8,084,056 and published PCT application WO99/36104 relating to the method of generating a lipid blend areincorporated by reference herein.

The organic solvent dissolution-precipitation process is preferred overthe aqueous suspension/lyophilization process for a number of reasons asoutlined in U.S. Pat. No. 8,084,056 and published PCT application WO99/36104, including the uniformly distributed lipid solid that resultsusing the organic dissolution method.

Alternatively, the lipids may be provided as individual powders that aredissolved together or individually directly into propylene glycol,glycerol or propylene glycol/glycerol to form the non-aqueous mixture.

As used herein, a lipid solution is a solution comprising a mixture oflipids. Similarly a lipid formulation is a formulation comprising one ormore lipids. The lipids may be cationic, anionic or neutral lipids. Thelipids may be of either natural, synthetic or semi-synthetic origin,including for example, fatty acids, fluorinated lipids, neutral fats,phosphatides, oils, fluorinated oils, glycolipids, surface active agents(surfactants and fluorosurfactants), aliphatic alcohols, waxes, terpenesand steroids.

At least one of the lipids may be a phospholipid, and thus the lipidblend may be referred to as a phospholipid blend. A phospholipid, asused herein, is a fatty substance containing an oily (hydrophobic)hydrocarbon chain (s) with a polar (hydrophilic) phosphoric head group.Phospholipids are amphiphilic. They spontaneously form boundaries andclosed vesicles in aqueous media.

Preferably all of the lipids are phospholipids, preferably1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC);1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA); and1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE). DPPA andDPPE may be provided as monosodium salt forms.

In some instances, the lipid components may be modified in order todecrease the reactivity of the microsphere with the surroundingenvironment, including the in vivo environment, thereby extending itshalf-life. Lipids bearing polymers, such as chitin, hyaluronic acid,polyvinylpyrrolidone or polyethylene glycol (PEG), may also be used forthis purpose. Lipids conjugated to PEG are referred to herein asPEGylated lipids. Preferably, the PEGylated lipid is DPPE-PEG orDSPE-PEG

Conjugation of the lipid to the polymer such as PEG may be accomplishedby a variety of bonds or linkages such as but not limited to amide,carbamate, amine, ester, ether, thioether, thioamide, and disulfide(thioester) linkages.

Terminal groups on the PEG may be, but are not limited to, hydroxy-PEG(HO-PEG) (or a reactive derivative thereof), carboxy-PEG (COOH-PEG),methoxy-PEG (MPEG), or another lower alkyl group, e.g., as iniso-propoxyPEG or t-butoxyPEG, amino PEG (NH2PEG) or thiol (SH-PEG).

The molecular weight of PEG may vary from about 500 to about 10000,including from about 1000 to about 7500, and from about 1000 to about5000. In some important embodiments, the molecular weight of PEG isabout 5000. Accordingly, DPPE-PEG5000 or DSPE-PEG5000 refers to DPPE orDSPE having attached thereto a PEG polymer having a molecular weight ofabout 5000.

The percentage of PEGylated lipids relative to the total amount oflipids in the lipid solution, on a molar basis, is at or between about2% to about 20%. In various embodiments, the percentage of PEGylatedlipids relative to the total amount of lipids is at or between 5 molepercent to about 15 mole percent.

Preferably, the lipids are 1,2-dtpalmttoyl-sn-glycero-3-phosphatidylcholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphatidic, mono sodium salt (DPPA), andN-(polyethylene glycol 5000carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine,monosodium salt (PEG5000-DPPE). The polyethylene glycol 5000 carbamoylmay be methoxy polyethylene glycol 5000 carbamoyl. In some importantembodiments, the lipids may be one, two or all three of DPPA, DPPC andPEG5000-DPPE. PEG5000-DPPE may be MPEG5000-DPPE or HO-PEG5000-DPPE.

A wide variety of lipids, like those described in Unger et al. U.S. Pat.No. 5,469,854, may be used in the present process. Suitable lipidsinclude, for example, fatty acids, lysolipids, fluorinated lipids,phosphocholines, such as those associated with platelet activationfactors (PAF) (Avanti Polar Lipids, Alabaster, Ala.), including1-alkyl-2-acetoyl-sn-glycero 3-phosphocholines, and1-alkyl-2-hydroxy-sn-glycero 3-phosphocholines; phosphatidylcholine withboth saturated and unsaturated lipids, includingdioleoylphosphatidylcholine; dimyristoylphosphatidylcholine;dipentadecanoylphosphatidylcholine; dilauroylphosphatdylcholine;1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC);distearoylphosphatidylcholine (DSPC); anddiarachidonylphosphatidylcholine (DAPC); phosphatidylethanolamines, suchas dioleoylphosphatidylethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) anddistearoyl-phosphatidylethanolamine (DSPE); phosphatidylserine;phosphatidylglycerols, including distearoylphosphatidylglycerol (DSPG);phosphatidylinositol; sphingolipids such as sphingomyelin; glycolipidssuch as ganglioside GM1 and GM2; glucolipids; sulfatides;glycosphingolipids; phosphatidic acids, such as1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) anddistearoylphosphatidic acid (DSPA); palmitic acid; stearic acid;arachidonic acid; and oleic acid.

Other suitable lipids include phosphatidylcholines, such asdiolecylphosphatidylcholine, dimyristoylphosphatidylcholine,dipalmitoylphosphatidylcholine (DPPC), anddistearoylphosphatidylcholine; phosphatidylethanolamines, such asdipalmitoylphosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine andN-succinyl-dioleoylphosphatidylethanolamine; phosphatidylserines;phosphatidyl-glycerols; sphingolipids; glycolipids, such as gangliosideGM1; glucolipids; sulfatides; glycosphingolipids; phosphatidic acids,such as dipalmatoylphosphatidic acid (DPPA); palmitic fatty acids;stearic fatty acids; arachidonic fatty acids; lauric fatty acids;myristic fatty acids; lauroleic fatty acids; physeteric fatty acids;myristoleic fatty acids; palmitoleic fatty acids; petroselinic fattyacids; oleic fatty acids; isolauric fatty acids; isomyristic fattyacids; isopalmitic fatty acids; isostearic fatty acids; cholesterol andcholesterol derivatives, such as cholesterol hemisuccinate, cholesterolsulfate, and cholesteryl-(4′-trimethylammonio)-butanoate;polyoxyethylene fatty acid esters; polyoxyethylene fatty acid alcohols;polyoxyethylene fatty acid alcohol ethers; polyoxyethylated sorbitanfatty acid esters; glycerol polyethylene glycol oxystearate; glycerolpolyethylene glycol ricinoleate; ethoxylated soybean sterols;ethoxylated castor oil; polyoxyethylene-polyoxypropylene fatty acidpolymers; polyoxyethylene fatty acid stearates;12-(((7′-diethylaminocoumarin-3-yl)-carbonyl)-methylamino)-octadecanoicacid;N-[12-(((7′-diethylamino-coumarin-3-yl)-carbonyl)-methyl-amino)octadecanoyl]-2-amino-palmiticacid; 1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinyl-glycerol; and1-hexadecyl-2-palmitoyl-glycerophosphoethanolamine andpalmitoylhomocysteine; lauryltrimethylammonium bromide(lauryl-=dodecyl-); cetyltrimethylammonium bromide (cetryl-=hexadecyl-);myristyltrimethylammonium bromide (myristyl-=tetradecyl-);alkyldimethylbenzylammonium chlorides, such as wherein alkyl is aC.sub.12, C.sub.14 or C.sub.16 alkyl; benzyldimethyldodecylammoniumbromide; benzyldimethyldodecylammonium chloride,benzyldimethylhexadecylammonium bromide; benzyldimethylhexadecylammoniumchloride; benzyldimethyltetradecylammonium bromide;benzyldimethyltetradecylammonium chloride; cetyldimethylethylammoniumbromide; cetyldimethylethylammonium chloride; cetylpyridinium bromide;cetylpyridinium chloride;N-[1-2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA);1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP); and1,2-dioleoyl-e-(4′-trimethylammonio)-butanoyl-sn-glycerol (DOTB).

In some embodiments where DPPA, DPPC and DPPE are used, their molarpercentages may be about 77-90 mole % DPPC, about 5-15 mole % DPPA, andabout 5-15 mole % DPPE, including DPPE-PEG5000. Preferred ratios of eachlipid include those described in the Examples section such as a weight %ratio of 6.0 to 53.5 to 40.5 (DPPA:DPPC:MPEG5000-DPPE) or a mole % ratioof 10 to 82 to 8 (10:82:8) (DPPA:DPPC:MPEG5000-DPPE).

The lipid concentration in the non-aqueous mixtures intended for longterm, room temperature storage may vary depending on the embodiment. Insome instances, the lipid concentration may range from about 0.1 mg toabout 20 mg per mL of non-aqueous mixture, including about 0.9 mg toabout 10 mg per mL of non-aqueous mixture and about 0.9 mg to about 7.5mg per mL of non-aqueous mixture. In some embodiments, the lipidconcentration may range from about 0.94 mg to about 7.5 mg lipid per mLof non-aqueous mixture, including about 1.875 mg to about 7.5 mg lipidper mL of non-aqueous mixture, or about 3.75 mg to about 7.5 mg lipidper mL of non-aqueous mixture. In some instances, the lipidconcentration is about 0.94 mg to about 1.875 mg per mL of non-aqueousmixture, about 1.875 mg to about 3.75 mg per mL of non-aqueous mixture,or about 3.75 mg to about 7.5 mg of total lipid per mL of non-aqueousmixture.

As an example, the lipid concentration may range from about 0.1 mg toabout 10 mg lipid per mL of propylene glycol/glycerol (combined),including about 1 mg to about 5 mg lipid per mL of propyleneglycol/glycerol (combined). In some instances, the lipid concentrationis about 0.94 mg to about 3.75 mg lipid per mL of propyleneglycol/glycerol (combined).

As another example, the lipid concentration may range from about 0.1 mgto about 20 mg lipid per mL of propylene glycol, including about 1 mg toabout 10 mg lipid per mL of propylene glycol, or about 2 mg to about 7.5mg lipid per mL of propylene glycol, or about 3.75 mg to about 7.5 mglipid per ml of propylene glycol. In some embodiments, the lipidconcentration is about 1.875 mg to about 7.5 mg lipid per mL ofpropylene glycol, including about 3.75 mg to about 7.5 mg lipid per mLof propylene glycol.

As yet another example, the lipid concentration may range from about 0.1mg to about 20 mg lipid per mL of glycerol, including about 1 mg toabout 10 mg lipid per mL glycerol, or about 2 mg to about 7.5 mg lipidper mL of glycerol, or about 3.75 mg to about 7.5 mg lipid per ml ofglycerol. In some instances, the lipid concentration is about 1.875 mgto about 7.5 mg lipid per mL of glycerol, including about 3.75 mg toabout 7.5 mg lipid per mL of glycerol.

The ability to generate compositions of lipid-encapsulated gasmicrospheres that are still useful as ultrasound contrast agents usinglower amounts of lipid, as compared to marketed ultrasound contrastagent lipid formulations, is beneficial since it reduces the maximumamount of lipids (and other constituents) that could be administered toa subject from a single vial, thereby reducing the chance of accidentaloverdosing of a subject.

Propylene glycol is a liquid at room temperature having a density of1.035 g/ml at 20° C. Glycerol is a liquid at room temperature having adensity of 1.26 g/ml at 20° C.

The total volume of the non-aqueous mixtures capable of long term, roomtemperature storage may range depending on the final intended use. As anexample, volumes may range from about 0.05 to about 10 mL, or about 0.1to about 10 mL, or about 0.1 to about 5 mL, or about 0.25 to about 5 mL,or about 0.5 to about 1 mL, or about 0.1 to about 1.0 mL.

It is to be understood that these non-aqueous mixtures will typically bediluted, for example, with an aqueous solution prior to activation, asdescribed below, and/or prior to administration to a subject. Totaldilution may be about 1-fold to about 100-fold, including about 5-foldto about 30-fold, including about 5-fold, about 10-fold, about 20-fold,and about 50-fold.

In some embodiments, the lipid formulations comprising lipid, propyleneglycol and glycerol may be diluted about 5-fold prior to activation. Insome embodiments, the lipid formulations comprising lipid and propyleneglycol may be diluted about 10-fold prior to activation. In someembodiments, the lipid formulations comprising lipid and glycerol may bediluted about 10-fold prior to activation. Thereafter the dilutedcomposition may be further diluted by about 1-fold to about 50-fold,including about 10-fold to about 50-fold, including about 10-fold.

Accordingly, the afore-mentioned lipid, propylene glycol and glycerolconcentrations will change upon dilution. For example, in instanceswhere the dilution is about 10-fold, the lipid concentrations of thefinal formulation are about 10-fold reduced to those recited above.Similar reductions will occur in propylene glycol and/or glycerolconcentrations.

Gas

The non-aqueous mixtures may be provided with a gas. For example, thenon-aqueous mixtures may be provided in contact with a gas, or they maybe provided in the same container or housing as a gas but not in contactwith the gas (i.e., the non-aqueous mixture and the gas may bephysically separate from each other).

It was not heretofore known or expected that these non-aqueous mixturescould be stably stored long term, at room temperature in contact with agas such as a perfluorocarbon gas. It was also not known or expectedthat these non-aqueous mixtures could be activated to formlipid-encapsulated gas microspheres. It was further found in accordancewith the invention that certain of these non-aqueous mixtures can beused to form microspheres in sufficient number and of sufficient size(as expressed for example as diameter) to be clinically useful.

The gas is preferably substantially insoluble in the lipid formulationsprovided herein such as the non-aqueous mixture. The gas may be anon-soluble fluorinated gas such as sulfur hexafluoride or aperfluorocarbon gas. Examples of perfluorocarbon gases includeperfluoropropane, perfluoromethane, perfluoroethane, perfluorobutane,perfluoropentane, perfluorohexane. Examples of gases that may be used inthe microspheres of the invention are described in U.S. Pat. No.5,656,211 and are incorporated by reference herein. In an importantembodiment, the gas is perfluoropropane.

Examples of gases include, but are not limited to, hexafluoroacetone,isopropylacetylene, allene, tetrafluoroallene, boron trifluoride,1,2-butadiene, 1,3-butadiene, 1,2,3-trichlorobutadiene,2-fluoro-1,3-butadiene, 2-methyl-1,3 butadiene,hexafluoro-1,3-butadiene, butadiyne, 1-fluorobutane, 2-methylbutane,decafluorobutane (perfluorobutane), decafluoroisobutane(perfluoroisobutane), 1-butene, 2-butene, 2-methy-1-butene,3-methyl-1-butene, perfluoro-1-butene, perfluoro-1-butene,perfluoro-2-butene, 4-phenyl-3-butene-2-one, 2-methyl-1-butene-3-yne,butylnitrate, 1-butyne, 2-butyne,2-chloro-1,1,1,4,4,4-hexafluoro-butyne, 3-methyl-1-butyne,perfluoro-2-butyne, 2-bromo-butyraldehyde, carbonyl sulfide,crotononitrile, cyclobutane, methylcyclobutane, octafluorocyclobutane(perfluorocyclobutane), perfluoroisobutane, 3-chlorocyclopentene,cyclopropane, 1,2-dimethylcyclopropane, 1,1-dimethylcyclopropane, ethylcyclopropane, methylcyclopropane, diacetylene,3-ethyl-3-methyldiaziridine, 1,1,1-trifluorodiazoethane, dimethylamine,hexafluorodimethylamine, dimethylethylamine, bis-(dimethylphosphine)amine, 2,3-dimethyl-2-norbornane, perfluoro-dimethylamine,dimethyloxonium chloride, 1,3-dioxolane-2-one,1,1,1,1,2-tetrafluoroethane, 1,1,1-trifluoroethane,1,1,2,2-tetrafluoroethane, 1,1,2-trichloro-1,2,2-trifluoroethane,1,1-dichloroethane, 1,1-dichloro-1,2,2,2-tetrafluoroethane,1,2-difluoroethane, 1-chloro-1,1,2,2,2-pentafluoroethane,2-chloro-1,1-difluoroethane, 1-chloro-1,1,2,2-tetrafluoro-ethane,2-chloro-1,1-difluoroethane, chloroethane, chloropentafluoroethane,dichlorotrifluoroethane, fluoroethane, nitropentafluoroethane,nitrosopentafluoro-ethane, perfluoroethane, perfluoroethylamine, ethylvinyl ether, 1,1-dichloroethylene, 1,1-dichloro-1,2-difluoro-ethylene,1,2-difluoroethylene, methane, methane-sulfonyl-chlori-detrifluoro,methane-sulfonyl-fluoride-trifluoro, methane-(pentafluorothio)trifluoro,methane-bromo-difluoro-nitroso, methane-bromo-fluoro,methane-bromo-chloro-fluoro, methane-bromo-trifluoro,methane-chloro-difluoro-nitro, methane-chloro-dinitro,methane-chloro-fluoro, methane-chloro-trifluoro,methane-chloro-difluoro, methane-dibromo-difluoro,methane-dichloro-difluoro, methane-dichloro-fluoro, methane-difluoro,methane-difluoro-iodo, methane-disilano, methane-fluoro,methane-iodomethane-iodo-trifluoro, methane-nitro-trifluoro,methane-nitroso-triofluoro, methane-tetrafluoro,methane-trichloro-fluoro, methane-trifluoro, methanesulfenylchloride-trifluoro, 2-methyl butane, methyl ether, methylisopropyl ether, methyl lactate, methyl nitrite, methyl sulfide, methylvinyl ether, neopentane, nitrogen (N.sub.2), nitrous oxide,1,2,3-nonadecane tricarboxylic acid-2-hydroxycrimethylester,1-nonene-3-yne, oxygen (O.sub.2), oxygen 17 (.sup.17 O.sub.2),1,4-pentadiene, n-pentane, dodecafluoropentane (perfluoropentane),tetradecafluorohexane (perfluorohexane), perfluoroisopentane,perfluoroneopentane, 2-pentanone-4-amino-4-methyl, 1-pentene, 2-pentene{cis}, 2-pentene {trans}, 1-pentene-3-bromo, 1-pentene-perfluoro,phthalic acid-tetrachloro, piperidine-2,3,6-trimethyl, propane,propane-1,1,1,2,2,3-hexafluoro, propane-1,2-epoxy, propane-2,2 difluoro,propane-2-amino, propane-2-chloro, propane-heptafluoro-1-nitro,propane-heptafluoro-1-nitroso, perfluoropropane, propene,propyl-1,1,1,2,3,3-hexafluoro-2,3 dichloro, propylene-1-chloro,propylene-chloro-{trans}, propylene-2-chloro, propylene-3-fluoro,propylene-perfluoro, propyne, propyne-3,3,3-trifluoro, styrene-3-fluoro,sulfur hexafluoride, sulfur (di)-decafluoro(S.sub.2 F.sub.10),toluene-2,4-diamino, trifluoroacetonitrile, trifluoromethyl peroxide,trifluoromethyl sulfide, tungsten hexafluoride, vinyl acetylene, vinylether, neon, helium, krypton, xenon (especially rubidium enrichedhyperpolarized xenon gas), carbon dioxide, helium, and air.

Fluorinated gases (that is, a gas containing one or more fluorinemolecules, such as sulfur hexafluoride), fluorocarbon gases (that is, afluorinated gas which is a fluorinated carbon or gas), andperfluorocarbon gases (that is, a fluorocarbon gas which is fullyfluorinated, such as perfluoropropane and perfluorobutane) arepreferred.

The gas such as the perfluorocarbon gas is typically present below itsordinary concentration at room temperature due to the incorporation ofair during production. The concentration of perfluoropropane whenpresent in a vial comprising a non-aqueous mixture and a gas headspaceis expected to be about 6.52 mg/mL, at about one atmosphere of pressure.The concentrations of other gases, as known in the art, would besimilarly diluted due to incorporation of air during production.

The invention contemplates that the non-aqueous mixtures providedherein, whether in contact with or physically separate from a gas suchas a perfluorocarbon gas, may be stored at a temperature in the range ofabout 4° C. to about 40° C., about 4° C. to about 30° C., about 4° C. toabout 25° C., about 10° C. to about 40° C., about 15° C. to about 40°C., or about 15° C. to about 30° C.

The invention further contemplates that the non-aqueous mixturesprovided herein, whether in contact with or physically separate from agas such as a perfluorocarbon gas, may be stored for about 1 month toabout 6 months, about 1 month to about 1 year, or about 1 month to about2 years. Thus, the non-aqueous mixtures provided herein, whether incontact with or physically separate from a gas such as a perfluorocarbongas, may be stored for about 1 month to about 2 years at a temperaturerange of about 15° C. to about 30° C., as a non-limiting example.

Containers and Chamber Configurations

The non-aqueous mixtures may be provided in a container (or housing).The container may be a single chamber or a multi-chamber container, suchas but not limited to a dual chamber container.

In some embodiments, the container is a vial. The vial may be made ofany material including but not limited to glass or plastic. The glassmay be pharmaceutical grade glass. The container may be sealed with astopper such as a rubber stopper. In some embodiments, the container isa 0.5-10 mL container. The container may be a 1-5 mL container, or a 1or 2 mL container. Such volumes refer to the volume of liquid typicallyplaced into the container (referred to as the liquid fill volume). Thisis in contrast to the entire internal volume of the container, whichwill be higher than the liquid fill volume. Examples of liquid fill andinternal volumes are as follows: Schott 2 mL (liquid fill volume) vialhaving a 2.9 mL internal volume; Schott 3 mL (liquid fill volume) vialhaving a 4.5 mL internal volume; and Wheaton 1 mL (liquid fill volume)v-vial having a 1.2 mL internal volume.

As will be understood in the context of this disclosure, the internalvolume of a container may be occupied with non-aqueous mixture and gas.An example of a suitable container is the Wheaton 2 ml glass vial(commercially available from, for example, Nipro, Cat. No. 2702, B33BA,2 cc, 13 mm, Type I, flint tubing vial), having an actual internalvolume of about 3.75 ml. An example of a suitable stopper is a West graybutyl lyo, siliconized stopper (Cat. No. V50, 4416/50, 13 mm). Anexample of a suitable seal is a West flip-off aluminum seal (Cat. No.3766, white, 13 mm). The containers are preferably sterile and/or aresterilized after introduction of the lipid solution and/or gas asdescribed in published PCT application WO99/36104.

In some embodiments, the container is a flat bottom container such as aflat-bottom vial. Suitable vials include flat bottom borosilicate vials,including Wheaton vials. In some embodiments, the container is anon-flat bottom container or vial. In some embodiments, the container isa V-bottom container such as a V-bottom vial. In some embodiments, thecontainer is a round-bottom container such as round-bottom vial. In someembodiments, the container has converging walls such that its bottomsurface area (or bottom surface diameter) is smaller than its top(opening) surface area (or diameter) or smaller than any diametertherebetween (e.g., a body diameter). For clarity, a V-bottom containeror vial has converging walls, and its bottom surface area issignificantly smaller than any of it top or body surface areas.

In some embodiments, the container is a syringe. The non-aqueous mixturemay be provided in a pre-filled syringe, optionally in physical contactwith the gas.

In some embodiments, the container is a single chamber container, suchas a vial. In such a single chamber, the non-aqueous mixture and thegas, if present, may be in physical contact with each other.

In some embodiments, the containers comprise two or more chambers. Thecontents of the two chambers are physically separated from each other,for example during storage. However, when used, the contents of the twochambers are combined and intermingled. Thus, the container furthercomprises a barrier that physically separates the contents of the firstand second chambers but that can be “removed” in order to combine thosecontents ultimately. The disclosure contemplates any possible means ofremoving such barrier including pressure, mechanical piercing orpunching, dissolution, and the like.

Dual chamber devices such as dual chamber syringes or dual chamber tubesare known in the art and are commercially available. Non-limitingexamples include Vetter dual chamber syringes and NeoPak dual chambertubes.

In some embodiments, a non-aqueous mixture consisting of or consistingessentially of one or more lipids, propylene glycol, or glycerol, orpropylene glycol/glycerol, and a non-phosphate buffer is provided in acontainer such as a single chamber container. Such a mixture may beprovided with or without a gas such as a perfluorocarbon gas. Ifprovided with the gas, the gas may be in the same chamber as thenon-aqueous mixture or in a separate chamber of a multi-chambercontainer, as provided below.

The container may have two chambers, wherein a first chamber comprisesthe non-aqueous mixture comprising the lipid(s) such as DPPA, DPPC andPEG5000-DPPE in propylene glycol and glycerol or propylene glycol orglycerol, and a second chamber comprises a gas such as a perfluorocarbongas. The non-aqueous mixture may comprise a buffer such as anon-phosphate buffer.

In another embodiment, the container may have two chambers, wherein afirst chamber comprises

(i) the non-aqueous mixture comprising

-   -   (a) the lipid(s) such as DPPA, DPPC and PEG5000-DPPE and    -   (b) propylene glycol and glycerol or propylene glycol or        glycerol, and

(ii) a gas such as a perfluorocarbon gas, and a second chamber comprisesan aqueous diluent.

The non-aqueous mixture may comprise a buffer such as a non-phosphatebuffer. Alternatively, the aqueous solution may comprise a buffer suchas a phosphate buffer.

In another embodiment, the container may have two chambers, wherein afirst chamber comprises

(i) the non-aqueous mixture comprising

-   -   (a) the lipid(s) such as DPPA, DPPC and PEG5000-DPPE and    -   (b) propylene glycol and glycerol or propylene glycol or        glycerol, and a second chamber comprises

(i) an aqueous diluent, and

(ii) a gas such as a perfluorocarbon gas.

In another embodiment, the container may have at least three chambers,wherein a first chamber comprises a non-aqueous mixture comprising DPPA,DPPC and PEG5000-DPPE in propylene glycol or glycerol or propyleneglycol and glycerol, a second chamber comprises a gas such as aperfluorocarbon gas, and a third chamber comprises an aqueous solution.

In another embodiment, the container may comprise a first chamber thatcomprises the non-aqueous mixture comprising lipids and propylene glycoland a second chamber that comprises glycerol. In another embodiment, thecontainer may comprise a first chamber that comprises the non-aqueousmixture comprising lipids and glycerol and a second chamber thatcomprises propylene glycol.

The aqueous diluent may comprise salts such as but not limited to sodiumchloride, and thus may be regarded as a saline solution. The aqueousdiluent may comprise a buffer such as a phosphate buffer, and thus maybe regarded as a buffered aqueous diluent. The aqueous diluent may be abuffered saline solution. The non-aqueous mixture may comprise a buffersuch as a non-phosphate buffer, examples of which are provided herein.The non-aqueous mixture and the aqueous diluent may both comprise abuffer. In typical embodiments, either the non-aqueous mixture or theaqueous diluent comprises a buffer, but not both. The bufferconcentration will vary depending on the type of buffer used, as will beunderstood and within the skill of the ordinary artisan to determine.The buffer concentration in the non-aqueous lipid formulation may rangefrom about 1 mM to about 100 mM. In some instances, the bufferconcentration may be about 1 mM to about 50 mM, or about 1 mM to about20 mM, or about 1 mM to about 10 mM, or about 1 mM to about 5 mM,including about 5 mM.

The final formulation to be administered typically intravenously to asubject including a human subject may have a pH in the range of 4-8 orin a range of 4.5-7.5. In some instances, the pH may be in a range ofabout 6 to about 7.5, or in a range of 6.2 to about 6.8. In still otherinstances, the pH may be about 6.5 (e.g., 6.5+/−0.5 or +/−0.3). In someinstances, the pH may be in a range of 5 to 6.5 or in a range of 5.2 to6.3 or in a range of 5.5 to 6.1 or in a range of 5.6 to 6 or in a rangeof 5.65 to 5.95. In still another instance, the pH may be in a range ofabout 5.7 to about 5.9 (e.g., +/−0.1 or +/−0.2 or +/−0.3 either or bothends of the range). In another instance, the pH may be about 5.8 (e.g.,5.8+/−0.15 or 5.8+/−0.1).

In some embodiments, the aqueous diluent comprises glycerol, a buffersuch as phosphate buffer, salt(s) and water. Such an aqueous diluent maybe used with a non-aqueous mixture that lacks glycerol. In someembodiments, the lipid solution further comprises saline (salt(s) andwater combined) and glycerol in a weight ratio of 8:1.

In some embodiments, the aqueous diluent comprises propylene glycol, abuffer such as phosphate buffer, salt(s) and water. Such an aqueousdiluent may be used with a non-aqueous mixture that lacks propyleneglycol.

In some embodiments, the aqueous diluent comprises a buffer such asphosphate buffer, salt(s) and water. Such an aqueous diluent may be usedwith a non-aqueous mixture that comprises both propylene glycol andglycerol.

Provided herein is a method comprising placing a non-aqueous mixture oflipids and propylene glycol, and a gas into a container, a methodcomprising placing a non-aqueous mixture of lipids and glycerol, and agas into a container, and a method comprising placing a non-aqueousmixture of lipids and propylene glycol and glycerol, and a gas into acontainer. In any of these methods, the gas may be placed into thecontainer through exchange of the headspace gas. Gas exchangers suitablefor this purpose are known in the art. An example of a gas exchangedevice is a lyophilizing chamber. Such containers may then be stored atabout 10 to about 50° C., or about 15 to about 40° C., or about 20 toabout 30° C. for up to 2 years, or for 1 to 12 months, or for 1-30 days.In another aspect, the container may be provided with instructions forstorage at the foregoing temperatures, optionally for the foregoingperiods of time, or alternatively lacking instructions for storage at 4°C. or under refrigeration.

Provided herein is a method comprising combining a first compositioncomprising a non-aqueous solution of lipids in propylene glycol andperfluorocarbon gas with a second composition comprising an aqueousdiluent, a method comprising combining a first composition comprising anon-aqueous solution of lipids in glycerol and perfluorocarbon gas witha second composition comprising an aqueous diluent, and a methodcomprising combining a first composition comprising a non-aqueoussolution of lipids in propylene glycol and glycerol and perfluorocarbongas with a second composition comprising an aqueous diluent.

The first and second compositions may be provided in first and secondchambers of a container, respectively, and combining may comprisebreaking a seal between the first and second chambers. The firstcomposition may be provided in a vial and the second composition may beprovided in a syringe, with the contents of the syringe being added tothe contents of the vial. Alternatively, the second composition may beprovided in a vial and the first composition may be provided in asyringe, with the contents of the syringe being added to the contents ofthe vial.

It is to be understood that any combination or variation on theforegoing embodiments is contemplated and embraced by this disclosure,and that the foregoing examples are not to be considered limiting unlessexpressly indicated.

Any of the foregoing container embodiments may be provided, with orwithout an additional housing, with instructions for storage at atemperature above 4° C. (or without refrigeration) or with instructionsthat are silent regarding storage temperature. It is to be understoodthat the formulations provided herein may be stored at 4° C. but thereis no requirement that they be stored at this temperature. Theinstructions may further recite long term storage such as storage fordays, months or even years and may further recite that long term storageoccur at or about room temperature (e.g., 18-25° C.).

In some embodiments, the composition is in a container, such as a vial,and such container is labeled. The container may have a label affixed toone or more of its outer surfaces. Such label may be a paper label orother such label that is visible by eye and capable of being read andunderstood by an end user without further aid or device. Alternatively,the label may be one that is machine- or device readable. Examples ofmachine- or device-readable labels include magnetic stripes, chips,barcodes including linear, matrix and 2D barcodes, radio frequencyidentification (RFID) tags, and the like. Barcodes such as linearbarcodes may be those that comply with or meet Uniform Code Councilstandards or Health Industry Business Communications Council standards.Such labels may in turn be read, for example, from a device such as amagnetic stripe reader, a chip reader, a barcode scanner or reader, anRFID tag reader, and the like. Virtually any labeling technology thathas been used for authentication and/or “track and trace” purposes maybe used in conjunction with the containers provided herein.

The label may provide the end user or an intermediate handler of thecontainer a variety f information including but not limited to sourceand/or producer of the composition contained therein, including forexample the name of the company or company subsidiary that made thecomposition and/or that produced components of the composition, the dateon which the composition was made, the physical location where thecomposition was made, the date of shipment of the container, thetreatment of the container including for example whether it was storedin a remote location and the conditions and length of such storage, thedate on which the container was delivered, the means of delivery, theNational Drug Code (NDC) as prescribed by the FDA, content of thecontainer, dose and method of use including route of administration,etc.

The label may serve one or more purposes including for exampleauthentication of the container and the composition contained therein.Authentication means the ability to identify or mark the container asoriginating and having been made by an authorized party, and it allowsan end user or other party to identify container and compositionsoriginating from another, unauthorized party. The label may also be usedto track and trace a container. This feature can be used to follow acontainer and the composition contained therein following production andup to the point of administration to a subject. In this regard, themovement of the container during that period of time may be stored in adatabase, and optionally such database may be accessible to an end userto ensure the integrity of the composition.

The label may also be a combined label, intending that it may containinformation that is read using two different modes. For example, thelabel may contain information that is apparent and understandable to thevisible eye (e.g., it may recite the name of the producer in words) andother information that is machine-readable, such as RFID embedded orbarcode embedded information.

The label may also be a dual use label, intending that it may serve twoor more purposes. For example, the label may contain information thatidentifies the composition and further information that identifies themanufacturer and/or date of manufacture. This information may beconveyed in the same format or using different format (e.g., one may beprovided in an RFID label and the other may be provided in a barcodelabel).

The label may provide content that is visible and understandable to ahuman, such as for example the name of the manufacturer. Alternativelyor additionally, the label may contain information that while readilyvisible to the human eye nevertheless provides no meaningful informationin the absence of a lookup table or other form of database to whichreference must be made. Such information for example may be provided asalpha-numeric code.

Activation

Any of the foregoing compositions may be used to form lipid-encapsulatedgas microspheres which in turn can be used as an ultrasound contrastagent. As used herein, lipid-encapsulated gas microspheres are sphereshaving an internal volume that is predominantly gas and that isencapsulated by a lipid shell. The lipid shell may be arranged as aunilayer or a bilayer, including unilamellar or multilamellar bilayers.These microspheres are useful as ultrasound contrast agents.

Microspheres are generated from the non-aqueous mixtures through aprocess of activation. Activation, as described in greater detailherein, refers to a vigorous shaking of a lipid solution (such as anon-aqueous solution) for the purpose of producing lipid-encapsulatedgas microspheres. Activation typically produces at least 1×10⁷microspheres per ml of solution, 5×10⁷ microspheres per ml of solution,or at least 7.5×10⁷ microspheres per ml of solution, or at least 1×10⁸microspheres per ml of solution, or about 1×10⁹ microspheres per ml ofsolution.

The disclosure contemplates that certain non-aqueous mixtures providedherein can be used to form lipid-encapsulated gas microspheres in thepresence of a gas. It was unexpected that these non-aqueous mixturescould be activated.

Activation may be performed by vigorous agitation including shaking fora defined period of time. As described above, activation may occur inthe presence or absence of an aqueous diluent. Activation, as usedherein, is defined as a motion that agitates a lipid solution such thata gas is introduced from the headspace into the lipid solution. Any typeof motion that agitates the lipid solution and results in theintroduction of gas may be used for the shaking. The agitation must beof sufficient force to allow the formation of foam after a period oftime. Preferably, the agitation is of sufficient force such that foam isformed within a short period of time, such as 30 minutes, and preferablywithin 20 minutes, and more preferably, within 10 minutes. In someembodiments, activation can occur in less than 5 minutes, less than 4minutes, less than 3 minutes, less than 2 minutes, in about 75 seconds,less than a minute, or in about 45 seconds. The agitation may be bymicroemulsifying, by microfluidizing, for example, swirling (such as byvortexing), side-to-side, or up and down motion. Different types ofmotion may be combined. The agitation may occur by shaking the containerholding the lipid solution, or by shaking the lipid solution within thecontainer without shaking the container itself. Further, the shaking mayoccur manually or by machine. Mechanical shakers that may be usedinclude, for example, a shaker table, such as a VWR Scientific(Cerritos, Calif.) shaker table, a microfluidizer, Wig-L-Bug™ (CrescentDental Manufacturing, Inc., Lyons, Ill.), and a mechanical paint mixer,VIALMIX®, or any of the devices described in Example 12 Vigorous shakingis defined as at least about 60 shaking motions per minute. This ispreferred in some instances. Vortexing at at least 1000 revolutions perminute is an example of vigorous shaking and is more preferred in someinstances. Vortexing at 1800 revolutions per minute is even morepreferred in some instances.

VIALMIX® is described in U.S. Pat. No. 6,039,557. Containers such asvials may be sufficiently agitated using VIALMIX® for the ranges oftimes recited above, including for example 45 seconds. Activation usingVIALMIX® may occur for less than 1 minute or longer, including for 30seconds, 45 seconds, 60 seconds, 75 seconds, 90 seconds, 105 seconds,120 seconds or longer.

Further examples of activation methods are provided in Example 12.

Non-aqueous mixtures comprising lipids and propylene glycol and glycerolmay be activated in the presence of a gas without addition of othersolutions. Alternatively, this mixture may be first combined with anaqueous diluent, and then activated in the presence of a gas.

Non-aqueous mixtures comprising of lipids and propylene glycol may befirst combined with glycerol, and optionally an aqueous diluent, andthen activated in the presence of a gas.

Non-aqueous mixtures comprising lipids and glycerol may be firstcombined with propylene glycol, and optionally an aqueous diluent, andthen activated in the presence of a gas.

In other instances, the lipids in solid form, whether as a lipid blendor not, may be dissolved in propylene glycol alone or glycerol alone orin propylene glycol and glycerol or in propylene glycol, glycerol and anaqueous diluent that may in turn comprise salt(s) and buffer. Any one ofthese mixtures may be activated, and in some instances, may be furtherdiluted with an aqueous diluent, prior to use.

Thus provided herein is a composition comprising lipid-encapsulated gasmicrospheres comprising DPPA, DPPC and PEG5000-DPPE in a non-aqueousmixture comprising propylene glycol and glycerol and a perfluorocarbongas, a composition comprising lipid-encapsulated gas microspherescomprising DPPA, DPPC and PEG5000-DPPE in a non-aqueous mixturecomprising propylene glycol and a perfluorocarbon gas, and a compositioncomprising lipid-encapsulated gas microspheres comprising DPPA, DPPC andPEG5000-DPPE in a non-aqueous mixture comprising glycerol and aperfluorocarbon gas.

The disclosure also contemplates formation of the microspheres in thepresence of an aqueous diluent such as but not limited to an aqueousbuffered saline solution. The aqueous diluent may comprise salt(s),buffer(s), propylene glycol, glycerol and water.

In some embodiments, an activated composition comprisinglipid-encapsulated gas microspheres may comprise saline, glycerol andpropylene glycol is a weight % ratio of 8:1:1.

Once formed, the microspheres may be diluted in an aqueous diluent, andthen administered to a subject. The aqueous saline solution willtypically be pharmaceutically acceptable and may lack preservatives(being referred to herein as preservative-free). The aqueous diluent maybe a saline solution (i.e., it may contain salt such as but not limitedto sodium chloride) and/or it may contain a buffer such as but notlimited to a phosphate buffer. The lipid-encapsulated gas microspheresmay be diluted by about 5 to about 50 fold, or about 35 to about 45fold. The diluted lipid-encapsulated gas microspheres may beadministered by bolus or continuous infusion into a subject in need ofultrasound contrast imaging.

The microspheres have an average diameter in the micron range. In someembodiments, the microspheres have an average diameter ranging fromabout 1.0 to about 2.0 microns, or about 1.2 microns to about 1.8microns. In some embodiments, the microspheres have an average diameterof about 1.6 microns.

In some embodiments, a majority of the microspheres may have a diameterin the range of about 1.0 to about 3.0 microns, or about 1.0 to about2.0 microns, or about 1.2 to about 2.0 microns, preferably in the rangeof about 1.2 to about 1.8 microns. The majority of microspheres means atleast 50%, preferably at least 75%, more preferably at least 80%, andeven more preferably at least 90% of the measured lipid-encapsulated gasmicrospheres in the composition. In some embodiments, at least 50%, orat least 60%, or at least 70%, or at least 80%, at least 90%, or atleast 95% of the detected lipid-encapsulated gas microspheres in thecomposition have a diameter in any of the foregoing ranges.

An average diameter represents the average diameter of all detectedmicrospheres in a composition. Microsphere diameter is typicallymeasured using instrumentation known and available in the art includingbut not limited to a Malvern FPIA-3000 Sysmex particle sizer. As will beunderstood in the art, such instrumentation typically has cutoff sizesfor both the lower and upper limits. This means that microspheres belowor above these cutoffs, respectively, are not counted (and are notincluded in the microsphere concentration calculation) and theirdiameter is not measured (and is not taken into consideration indetermining the average diameter of microspheres). The instrumentationused in the Examples had a 1.0 micron lower limit cutoff and a 40.0micron upper limit cutoff. The majority of counted or detectedmicrospheres, using a lower cutoff of 1.0 micron and an upper cutoff of40.0 microns, have a diameter in the range of 1.0 to 20.0 microns. It isto be understood that this disclosure uses the terms microsphere sizeand microsphere diameter interchangeably. Thus, unless otherwisespecified, microsphere size refers to microsphere diameter.

The composition provided herein including the activated compositions mayfurther comprise other constituents such as stabilizing materials oragents, viscosity modifiers, tonicity agents, coating agents, andsuspending agents. Examples of each class of agents are known in the artand are provided in for example U.S. Pat. No. 5,656,211, in publishedPCT application WO99/36104, and in published US application US2013/0022550.

The composition provided herein including the activated compositions maycomprise one or more buffers including but not limited to acetatebuffer, benzoate buffer, salicylate buffer, and/or phosphate buffer.

The pH of the composition may be about 6.2 to about 6.8. In someinstances, the pH may be in a range of 5 to 6.5 or in a range of 5.2 to6.3 or in a range of 5.5 to 6.1 or in a range of 5.6 to 6 or in a rangeof 5.65 to 5.95. In still another instance, the pH may be in a range ofabout 5.7 to about 5.9 (e.g., +/−0.1 or +/−0.2 or +/−0.3 either or bothends of the range). In another instance, the pH may be about 5.8 (e.g.,5.8+/−0.15 or 5.8+/−0.1). Such ranges may be achieved, for example,using an acetate buffered formulation diluted in water.

In some embodiments, each ml of the final composition (followingdilution of the non-aqueous solution with an aqueous diluent) comprises0.75 mg of lipids (consisting of 0.045 mg DPPA, 0.401 mg DPPC, and 0.304mg DPPE-PEG5000), 103.5 mg propylene glycol, 126.2 mg glycerol, 2.34 mgsodium phosphate monobasic monohydrate, 2.16 mg sodium phosphate dibasicheptahydrate, and 4.87 mg sodium chloride in water.

In some embodiments, each ml of final composition comprises about 0.43mg of lipids (consisting of 0.0225 mg DPPA, 0.2 mg DPPC, and 0.152 mgDPPE-PEG5000), 103.5 mg propylene glycol, 126.2 mg glycerol, 2.34 mgsodium phosphate monobasic monohydrate, 2.16 mg sodium phosphate dibasicheptahydrate, and 4.87 mg sodium chloride in water.

In some embodiments, each ml of the final composition (followingdilution of the non-aqueous solution with saline) comprises 0.75 mg oflipids (consisting of 0.045 mg DPPA, 0.401 mg DPPC, and 0.304 mgDPPE-PEG5000), 103.5 mg propylene glycol, 126.2 mg glycerol, 0.074 mgsodium acetate, 0.006 mg acetic acid, and 7.20 mg sodium chloride inwater.

Impurities and Stability

The invention further provides a method for assessing impurity contentin a lipid solution such as a non-aqueous solution. Such a methodcomprises analyzing a lipid solution for the presence of impuritiesusing any of a number of analytical methods such as but not limited tocharged aerosol detection (CAD) optionally coupled with one or moreseparation techniques such as HPLC. The lipid solution may be anon-aqueous solution comprising lipids and propylene glycol or glycerolor propylene glycol and glycerol. The lipid solution may furthercomprise a buffer such as a non-phosphate buffer. The lipid solution mayfurther comprise salt(s) and/or water. The presence of an impurity abovea threshold level may signify that the lipid solution was not storedproperly, that its stability has been compromised, and thus that thelipid solution should be discarded and not administered to a subject.Such a method could be used for quality control purposes.

Example 2 provides a method for measuring impurity content in anon-aqueous solution. The impurity content is provided as a % impurityrelative to the input (or theoretical or nominal) lipid amount, meaningthe impurity is expressed as a percentage of the total amount of lipidpresent assuming no loss of lipid.

The modified lipid formulations may comprise less than 10%, less than5%, or less than 2% impurities when stored at room temperature for aperiod of time, including for example, about 1 month, about 2 months,about 3 months, about 6 months, or longer including about 1 year, orabout 2 years.

Significantly, the modified lipid formulations may comprise fewerimpurities than DEFINITY® when both formulations are stored at roomtemperature (i.e., when the composition and DEFINITY® are stored at roomtemperature). This reduction in impurity level may be a difference ofabout 1%, about 2%, about 3%, about 4%, or about 5%, or more.

Uses and Applications

The invention provides methods of use of the microspheres andmicrosphere compositions. The microspheres are intended as ultrasoundcontrast agents, and they may be used in vivo in human or non-humansubjects or in vitro. The compositions of the invention may be used fordiagnostic or therapeutic purposes or for combined diagnostic andtherapeutic purposes.

When used as ultrasound contrast agents for human subjects, thecompositions are activated as described herein in order to form asufficient number of microspheres, optionally diluted into a largervolume, and administered in one or more bolus injections or by acontinuous infusion. Administration is typically intravenous injection.Imaging is then performed shortly thereafter. The imaging applicationcan be directed to the heart or it may involve another region of thebody that is susceptible to ultrasound imaging. Imaging may be imagingof one or more organs or regions of the body including withoutlimitation the heart, blood vessels, the cardiovasculature, and theliver.

Subjects of the invention include but are not limited to humans andanimals. Humans are preferred in some instances.

The lipid compositions are administered in effective amounts. Aneffective amount will be that amount that facilitates or brings aboutthe intended in vivo response and/or application. In the context of animaging application, such as an ultrasound application, the effectiveamount may be an amount of lipid microspheres that allow imaging of asubject or a region of a subject.

EXAMPLES Example 1. Sample Preparation

The commercial, FDA approved, ultrasound contrast agent, DEFINITY®(Lantheus Medical Imaging) was used for comparison. Each vial containsthe following: 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC;0.401 mg/mL), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA;0.045 mg/mL), and N-(methoxypolyethylene glycol 5000carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine(MPEG5000 DPPE; 0.304 mg/mL) in a matrix of 103.5 mg/mL propyleneglycol, 126.2 mg/mL glycerol, and 2.34 mg/mL sodium phosphate monobasicmonohydrate, 2.16 mg/mL sodium phosphate dibasic heptahydrate, and 4.87mg/mL sodium chloride in Water for Injection. The pH is 6.2-6.8. Thenominal fill volume of the lipid solution is approximately 1.76 mL in a2 cc Wheaton glass vial with an approximate volume of 3.80 mL and thus ahead space of approximately 2.04 mL containing perfluoropropane gas(PFP, 6.52 mg/mL).

New formulations were prepared as follows:

Lipid blend (LB) containing DPPC, DPPA, MPEG500 DPPE was prepared asdescribed in U.S. Pat. No. 8,084,056, the content of which are herebyincorporated by reference and may be used in the present process.Formulations of LB were prepared by mixing LB powder in propylene glycol(PG), or 1:1 v/v propylene glycol/glycerol (PG/G), or glycerol vehicleat 55° C. In some studies, 0.005 M acetate, benzoate, or salicylatebuffer prepared at salt to acid ratios of 90/10, 75/25, 50/50, 25/79 and10/90 were dissolved in the vehicle. In some instances, phosphate bufferwas included in an aqueous or saline solution.

Example 2. Lipid Stability

New formulation lipid blend samples from Example 1 in propylene glycolwere placed into 2 cc Wheaton glass vials, the headspace replaced withPFP gas, a West, grey butyl lyo stopper inserted and the vial crimpedwith an aluminum seal. Vials were stored in an environmental chamber at25° C. to represent room temperature storage or heated to 130° C. in adrying oven to represent terminal sterilization. At appropriate timepoints, sample vials were removed from storage, de-crimped, saline wasadded to the vial and mixed to ensure a homogenous solution. The samplewas transferred to a HPLC vial and analyzed by reverse phase HPLCseparation and Corona Charged aerosol detection (CAD; HPLC With ChargedAerosol Detection for the Measurement of Different Lipid Classes, I. N.Acworth, P. H. Gamache, R. McCarthy and D. Asa, ESA Biosciences Inc.,Chelmsford, Mass., USA; J. Waraska and I. N. Acworth, AmericanBiotechnology Laboratory, January 2008) for impurities.

Results for DEFINITY® vials stored 3 months at 25° C. are provided forcomparison. Analysis used gradient reverse phase HPLC with EvaporativeLight Scattering Detection, (ELSD) using a C18 column and mobile phasecontaining: water, methanol, ammonium acetate, and triethylamine. Tables1 and 2, provide the total impurity as a percentage of the total lipidcontent in the vial at 25° C. and 130° C.

TABLE 1 Impurity data for lipid blend (LB) in propylene glycol (PG)formulation stored at 25° C. DEFINITY ® 7.50 mg Lipid (0.75 mg LipidBlend/mL PG* Blend/mL) Number of 96 days 3 months Days at 25° C.(approximately 3 months) PERCENT TOTAL 2.1 11.86 IMPURITY *177 mg of PGcontaining LB (0.72 wt % LB; ratio of 1:138 for LB:PG).

TABLE 2 Impurity data for lipid blend (LB) in propylene glycol (PG)formulation and DEFINITY ® processed at 130° C. for 30 minutesDEFINITY ® 7.50 mg Lipid 3.75 mg Lipid (0.75 mg Lipid Blend/mL PG*Blend/mL PG** Blend/mL) PERCENT TOTAL 0.334 0.818 4.230 IMPURITY *89 mgof PG containing LB (0.72 wt % LB; ratio of 1:138 for LB:PG). **177 mgof PG containing LB (0.36 wt % LB; ration of 1:276 for LB:PG).

FIG. 1 illustrates the total impurity levels as a function of time forDEFINITY® at 2-8° C. and 25° C. and for the 7.5 mg LB/mL PG at 25° C.The total impurity level in DEFINITY® stored at 2-8° C. was similar tothat of the 7.5 mg LB/mL PG formulation stored at 25° C. When DEFINITY®was stored at 25° C., however, the level of total impuritiesdramatically increased.

These data demonstrate that the 7.5 mg LB/mL PG formulation is far morerobust than the DEFINITY® formulation at higher temperatures. Thisobservation was unexpected.

Example 3. Stability of Lipid Blend/Propylene Glycol/Glycerol (LB/PG/G)Formulation

New formulation lipid blend sample from Example 1 in 1:1 (v:v) propyleneglycol/glycerol was filled into a 2 cc Wheaton glass vial, the headspacereplaced with PFP gas, a West, grey butyl lyo stopper, inserted and thevial crimped with an aluminum seal. Vials were stored in anenvironmental chamber at 25° C. to represent room temperature storage orheated to 130° C. in an oven to represent terminal sterilization. Vialsstored at 25° C. were prepared and analyzed as described in Example 2.Vials heated at 130° C. were prepared as described in Example 2, butwere analyzed using the HPLC system as described for DEFINITY® inExample 2. Table 3 and 4, provide the total impurity as a percentage ofthe total lipid content in the vial at 25° C. and 130° C. Results forDEFINITY® analyzed as described in Example 2 are provided forcomparison.

TABLE 3 Impurity data for 3.75 mg lipid blend (LB) per mL in PG/Gformulation stored at 25° C. DEFINITY ® 3.75 mg Lipid (0.75 mg LipidBlend/mL PG/G* Blend/mL) Number of 87 days 3 months Days at 25° C.(approximately 3 months) PERCENT TOTAL 1.747 11.86 IMPURITY *391 mg ofPG/G containing LB (0.33 wt % LB:44.9 wt % PG:54.8 wt % G; ratio of1:138:168 for LB:PG:G).

TABLE 4 Impurity data for lipid blend (LB) in PG/G formulations andDEFINITY ® processed at 130° C. for 30 minutes DEFINITY ® 3.75 mg Lipid(0.75 mg Lipid Blend/mL PG/G* Blend/mL) PERCENT TOTAL 2.558 4.150IMPURITY *391 mg of PG/G containing LB (0.33 wt % LB:44.9 wt % PG:54.8wt % G; ratio of 1:138:168 for LB:PG:G).

FIG. 2 illustrates the total impurity levels as a function of time forDEFINITY® stored at 2-8° C. and 25° C. and for the 3.75 mg LB/mL PG/Gformulation stored at 25° C. The total impurity level in DEFINITY®stored at 2-8° C. was similar to that of the 3.75 mg LB/mL PG/Gformulation stored at 25° C. When DEFINITY® was stored at 25° C.,however, the level of total impurities dramatically increased.

These data demonstrate that the 3.75 mg LB/mL PG/G formulation is farmore robust than the DEFINITY® formulation at higher temperatures. Thisobservation was unexpected.

Example 4. Stability of Buffered Lipid Blend/Propylene Glycol/GlycerolFormulation

New formulation lipid blend sample from Example 1, in 1:1 (v:v)propylene glycol/glycerol containing 0.005M acetate (75/25 sodiumacetate/acetic acid), benzoate (75/25 sodium benzoate/benzoic acid) orsalicylate (90/10 sodium salicylate/salicylic acid) buffer was filledinto 2 cc Wheaton glass vial, the headspace replaced with PFP gas, aWest, grey butyl lyo stopper, inserted and the vial crimped with analuminum seal. Vials were stored at 25° C., prepared and analyzed asdescribed in Example 2. Results for DEFINITY® analyzed as described inExample 2 are provided for comparison. Table 5 provides the totalimpurity as a percentage of the total lipid content in the vial at 25°C.

FIG. 3 illustrates the total impurity levels as a function of time. WhenDEFINITY® was stored at 25° C., however, the level of total impuritiesdramatically increased. These data demonstrate that the 3.75 mg BufferedLB/mL PG/G formulation is far more robust than the DEFINITY® formulationat higher temperatures. This observation was unexpected.

TABLE 5 Impurity data for 3.75 mg Lipid Blend/mL Buffered PG/Gformulation stored at 25° C. 3.75 mg Lipid Blend/mL Buffered* PG/GFormulation DEFINITY ® 75/25 75/25 90/10 (0.75 mg Acetate BenzoateSalicylate Lipid Blend/mL) Number of 50 50 50 2 Months days at 25° C.Total Impurity 0.562 0.658 1.475 8.94 *5 mM buffer in 391 mg of PG/Gcontaining LB, (0.33 wt % LB; 44.9 wt % PG; 54.8 wt % G; ratio of1:138:168 for LB:PG:G). Ratios represent sodium acetate to acetic acid,sodium benzoate to benzoic acid, sodium salicylate to salicylic acid.

Example 5. Stability of Lipid Blend Glycerol Formulation

New formulation lipid blend sample from Example 1 in glycerol was filledinto 2 cc HPLC glass vial, the headspace replaced with PFP gas, and thevial sealed with a screw cap containing a septum. Vials were stored at25° C. and prepared and analyzed as described in Example 2.

Results for DEFINITY® analyzed as described in Example 2 are providedfor comparison. Table 6 provides the total impurity results for thisexperiment. These data demonstrate that the 7.5 mg buffered LB/mL Gformulation is far more robust than the DEFINITY® formulation at highertemperatures. This observation was unexpected.

TABLE 6 Impurity data for 7.50 mg Lipid Blend/mL glycerol (G)formulation* stored at 25° C. DEFINITY ® (Lot 4519M) 7.5 mg Lipid (0.75mg Lipid Blend/mL G Blend/mL) Number of 149 days 6 months Days at 25° C.(approximately 5 months) PERCENT TOTAL 2.478 23.17 IMPURITY *215 mg of Gcontaining LB, 0.59 wt % LB (ratio of 1:168 for LB:G).

Example 6. Stability of Lipid Blend Powder

LB powder was stored in an amber bottle with a PTFE lined cap at 25° C.Samples were prepared in a methanol (50%), propylene glycol (10%),glycerin (10%) ammonium acetate (30%, 5 mM) solution. The solution wastransferred to a HPLC vial and analyzed using a gradient reverse phaseHPLC with evaporative light scattering Detection, (ELSD) using a C18column and mobile phase containing: water, methanol, ammonium acetate,and triethylamine.

Table 7 provides stability data for lipid blend powder compared toDEFINITY® stored at 25° C.

These data demonstrate that the lipid blend powder is far more robustthan the DEFINITY® formulation at higher temperatures.

TABLE 7 Impurity Data for Lipid Blend Powder DEFINITY ® (0.75 mg LipidLB powder Blend/mL) 25° C. 25° C. Number of 87 days 3 months Days at 25°C. (approximately 3 months) PERCENT TOTAL 1.747 11.86 IMPURITY

Example 7. Activation of DEFINITY®

The commercially available, FDA approved, ultrasound contrast agent,DEFINITY® (Lantheus Medical Imaging, Inc.) is put into an active form(“activated”) by mechanical shaking (described in U.S. Pat. No.6,039,557, the content of which are hereby incorporated by reference andmay be used in the present process) of the PFP/lipid solution using aVIALMIX®. This results in incorporation of gas into lipid microspheresand represents the active product (see DEFINITY® prescribinginformation). Optimal VIALMIX® activation of DEFINITY® consistentlyproduces gas filled microspheres that can be analyzed for number andsize distribution using a particle sizer (Malvern FPIA-3000 Sysmex) whendiluted into an appropriate sheath solution (see Table 8) having lowerand upper cutoffs of 1 and 40 microns.

TABLE 8 DEFINITY ® bubble number and size analyzed using a MalvernFPIA-3000 Sysmex. Microsphere Microsphere DEFINITY ® Mean Diameter permL Sample (microns)^(a) (×10⁹)^(b) Sample 1 1.7 2.67 Sample 2 1.6 3.20Sample 3 1.7 3.20 Sample 4 1.7 1.75 Sample 5 1.6 2.77 Sample 6 1.6 2.97Average 1.7 2.76 ^(a)Mean microsphere diameter for microspheres rangingfrom 1 to 40 microns. ^(b)Mean microsphere concentration formicrospheres ranging from 1 to 40 microns.

Acoustic attenuation was measured for selected samples using a PhilipsSonos 5500 clinical ultrasound imaging system. Samples were diluted1:7.7 (1.3 ml plus 8.7 ml saline) in a 10 ml syringe. 200 microlitersamples from this syringe were pipetted into a beaker containing 200 mlof 0.9% saline at room temperature. A 2 cm stirring bar maintainedsolution uniformity and the s3 transducer of the ultrasound system waspositioned at the top of the beaker, just into the solution and 8.9 cmabove the upper margin of the stirring bar. 5 seconds of 120 Hz imageswere then acquired digitally and written to disk. The US system was usedin IBS mode, TGC was fixed at the minimal value for all depths, and LGCwas disabled. The mechanical index (MI) was 0.2 with power set 18 dBbelow maximum. The receive gain was fixed at 90 and the compression at0. For each sample tested US data acquisition was acquired prior to(blank) and after sample injection. Measurements were taken at 20, 60and 120 seconds after introduction of the sample into the beaker.

Image analysis was performed using Philips QLab, which read filesproduced by the US system and calculated values in dB for IBS mode.Regions of interest were drawn on the stirring bar and the dB valuesaveraged over the full 5 second (approximately 360 video frame)acquisition. Attenuation measurements were obtained by subtracting thesample ROI value from the blank ROI value (both in dB). This was dividedby twice the distance between the US transducer and the upper margin ofthe stirring bar to yield attenuation in dB/cm. Final values wereobtained by applying a linear regression of the samples taken withrespect to time after introduction to the beaker. The values used werederived from the intercept of the regression line with the y-axis.

TABLE 9 DEFINITY ® acoustic attenuation measurement^(a) Vial 1 Vial 2Vial 3 Mean SD DEFINITY ® 2.06 1.97 2.30 2.11 0.17 ^(a)The acousticattenuation of DEFINITY ® was determined using a Philips Sonos 5500.

Example 8. Activation of Non-Aqueous Formulations

New formulations of lipid blend described in Example 1 were weighed into2 cc Wheaton glass vials, diluent added if required, the headspacereplaced with PFP gas, a West, grey butyl lyo stopper, inserted and thevial crimped with an aluminum seal. Diluent was injected through thestopper if required, and the vial was mechanically shaken using VIALMIX®for a duration to produce optimum product activation. Microsphere numberand distribution was determined and some activated formulations wereexamined for ultrasound attenuation by the methods described in Example7.

TABLE 10 Microsphere characteristics for 7.5 mg lipid blend/mL PGformulation with formulation diluent^(a) added just prior to activation.Microsphere Microsphere Mean Diameter per mL Sample (microns) (×10⁹)LB/PG formulation 1.82 1.76 (add diluent then cap)^(b) LB/PG formulation1.82 1.92 (capped, then inject diluents through stopper)^(c)^(a)Formulation diluent contained glycerol, phosphate buffer and salineto match the DEFINITY ® vial composition upon dilution of formulation.^(b)177 mg propylene glycol formulation (0.72 wt % LB; ratio of 1:138for LB:PG), 1.59 mL diluent^(a) added, the headspace replaced with PFP,the 2 mL vial sealed with a West grey butyl stopper, crimped with analuminum seal, the vial activated for 45 sec with a VIALMIX ® and testedfor microsphere number and average size as described in Example 7.^(c)177 mg propylene glycol formulation (0.72 wt % LB; ratio of 1:138for LB:PG) in 2 mL vial, the headspace replaced with PFP and the vialcapped and crimped. Diluent^(a), 1.59 mL, was injected through thestopper into vial using a disposable syringe, the vial was immediatelyactivated for 45 sec with a VIALMIX ® and then tested as described inExample 7.

TABLE 11 Microsphere characteristics and acoustic attenuation for 7.5 mglipid blend/mL PG formulation with saline added just prior toactivation. Mean (SD) Microsphere Microsphere Acoustic Mean Diameter permL Attenuation^(b) Sample (microns) (×10⁹) (dB/cm) LB/PG formulation1.84 1.88 2.13 (0.34) (add saline then cap)^(a) ^(a)177 mg propyleneglycol formulation (0.72 wt % LB; ratio of 1:138 for LB:PG), 1.59 mL0.9% saline added, the headspace replaced with PFP, the 2 mL vial sealedwith a West grey butyl stopper, crimped with an aluminum seal, the vialactivated and tested for microsphere number and average size asdescribed in Example 7. ^(b)Acoustic attenuation determined as describedin Example 7.

TABLE 12 Microsphere characteristic and acoustic attenuation for 7.5 mglipid blend/mL PG/G formulations Mean Microsphere Microsphere Acoustic(SD) Mean Diameter per mL Attenuation^(c) Sample (microns) (×10⁹)(dB/cm) LB/PG/G 1.68 2.65 × 10⁹ Not formulation with determined salineadded followed by activation^(a) LB/PG/G 1.82 2.23 × 10⁹ 2.12 (0.27)formulation, activated and then diluted with saline^(b) ^(a)391 mgpropylene glycol and glycerol formulation (0.33% LB; 44.9% PG: 54.8% G;ratio of 1:138:168 for LB:PG:G), 1.38 mL 0.9% saline added, theheadspace replaced with PFP, the 2 mL vial sealed with a West grey butylstopper, crimped with an aluminum seal, the vial activated and testedfor microsphere number and average size as described in Example 7.^(b)391 mg propylene glycol and glycerol formulation (0.33% LB; 44.9%PG: 54.8% G; ratio of 1:138:168 for LB:PG:G), the headspace replacedwith PFP, and the vial capped and crimped as described in footnote aabove. Saline, 1.38 mL, was injected into vial using a disposablesyringe, the vial immediately activated and then tested as described inExample 7. ^(c)Acoustic attenuation determined as described in Example7.

TABLE 13 Microsphere characteristics for 7.5 mg lipid blend/mL buffered(5 mM) PG/G formulations with saline diluent added after activation.Sodium Acetate to Microsphere Microsphere Acetic Acid Ratio MeanDiameter per mL (5 mM total acetate) (microns) (×10⁹) 90:10 1.72 3.37 ×10⁹ 80:20 1.70 4.69 × 10⁹ 70:30 1.74 3.83 × 10⁹ 50:50 1.71 3.67 × 10⁹10:90 1.82 3.01 × 10⁹ ^(a) 391 mg buffered propylene glycol and glycerolformulation, (0.33% LB; 44.9% PG: 54.8% G; ratio of 1:138:168 forLB:PG:G), the 2 mL vial sealed with a West grey butyl stopper, crimpedwith an aluminum seal, the vial activated, 1.38 mL 0.9% saline added,the vial mixed and tested for microsphere number and average size asdescribed in Example 7.

These studies demonstrate that lipid blend formulated in a) PG b) PG/Gc) buffered PG/G can be activated to form microspheres that haveequivalent characteristics and acoustic attenuation to activatedDEFINITY® (as shown in Example 7) by simply adding diluent and shakingon a VIALMIX®. This demonstrates pre-formulation with aqueous diluent isnot required and simple addition is sufficient. Furthermore the diluentcan be added to the lipid formulation by injecting through the vialstopper. In addition the lipid blend in PG/G can be activated to formmicrospheres that have equivalent characteristics and acousticattenuation to activated DEFINITY® (as shown in Example 7) by shakingbefore the diluent is added. These findings are surprising.

Example 9. Activation of Individual Lipids or Lipid Blend

A formulation (Individual Lipid Formulation) was prepared by mixing theindividual phospholipids (DPPA, DPPC and MPEG5000 DPPE) in propyleneglycol at 0.045:0.401:0.304 (w:w:w) ratio (the same as the ratio forlipid blend). The resulting 7.5 mg/mL Individual lipid propylene glycolformulation was added to diluent (containing glycerol, phosphate bufferand saline to match the DEFINITY® vial composition) and mixed to form afinal total lipid concentration of 0.75 mg/mL. A 1.7 mL aliquot wasadded to a 2 cc Wheaton glass vial, the headspace replaced with PFP gas,a West, grey butyl lyo stopper, inserted and the vial crimped with analuminum seal. The vial was activated with a VIALMIX® and analyzed usingthe Sysmex FPIA 3000 for microsphere number and mean microsphere size.

TABLE 14 Microsphere characteristics for 7.5 mg individual lipid/mL PGformulation Microsphere Microsphere Mean Diameter per mL (microns)(×10⁹) Individual Lipid 1.7 2.46 Formulation

This study demonstrates that mixing individual lipids in PG, withoutpreparing a lipid blend, can produce a formulation that allows mixingwith a diluent to form a solution that can be activated to producemicrospheres with characteristics equivalent to activated DEFINITY®(when compared with Example 7).

In another experiment, lipid blend was weighed into a 2 cc Wheaton glassvial, matrix (PG/G/saline) was added to the vial, the headspace replacedwith PFP gas, a West, grey butyl lyo stopper, inserted, the vial crimpedwith an aluminum seal and then activated at 25° C. with a VIALMIX® andanalyzed using the Sysmex FPIA 3000 for microsphere number and meanmicrosphere size. The results are presented in Table 15.

TABLE 15 Microsphere characteristics for Lipid Blend Powder (1.275 mg)in a vial with PFP headspace Microsphere Microsphere Mean Diameter permL (microns) (×10⁹) Lipid Blend 1.63 4.10 Formulation

This study demonstrates that lipid blend powder could be weighed into avial, diluent added, headspace replaced with PFP, and the vial activatedto produce microspheres with characteristics equivalent to activatedDEFINITY® (when compared with Example 7). This demonstrates the lipidsdid not need to be pre-formulated to allow activation.

Example 10. Activation of Different Lipid Concentrations

Lipid blend, as described in Example 1, was used to make formulations atvarying total lipid blend concentrations by mixing different amounts ofLB powder in either propylene glycol (PG) or 1:1 v/v propyleneglycol/glycerol (PG/G). Each lipid formulation was weighed into 2 ccWheaton glass vials, diluent added if required, the headspace replacedwith PFP gas, a West, grey butyl lyo stopper, inserted and the vialcrimped with an aluminum seal. The vials were mechanically shaken usingVIALMIX® to activate the product and diluent added, if needed, throughthe stopper using a syringed equipped with a needle. Microsphere numberand distribution were determined as described in Example 7.

TABLE 16 Microsphere characteristics with different lipid mg/mL PGformulations^(a) Micro- Lipid Blend Lipid Blend sphere concentrationTime of concentration per mL Diameter in formulation Dilution afterdilution (×10⁹) (μm) DEFINITY ® n/a n/a 3.05 1.66 0.75 mg Lipid Blendper mL^(d) 7.5 mg Lipid Before  0.75 mg/mL 4.55 1.63 Blend/mL PGActivation^(b) 7.5 mg Lipid Before  0.75 mg/mL 4.65 1.72 Blend/mL PGActivation^(c) DEFINITY ® Before  0.375 mg/mL 1.38 1.66 diluted toactivation 0.375 mg Lipid Blend per mL^(d) 3.75 mg Lipid Before  0.375mg/mL 2.24 1.7 Blend/mL PG Activation^(b) 3.75 mg Lipid Before  0.375mg/mL 2.69 1.72 Blend/mL PG Activation^(c) DEFINITY ® Before 0.1875mg/mL 0.54 1.75 diluted to activation 0.1875 mg Lipid Blend per mL^(d)1.875 mg Lipid Before 0.1875 mg/mL 0.892 1.72 Blend/mL PG Activation^(b)1.875 mg Lipid Before 0.1875 mg/mL 1.25 1.74 Blend/mL PG Activation^(c)^(a)Vials (2 cc Wheaton vials) were prepared by weighting 177 mg ofpropylene glycol containing 1.875, 3.75 or 7.5 mg lipid blend/mL (ratiosof 1:552; 1:276; and 1:138 for LB:PG, respectively). ^(b)Vials werediluted with 8:1 (v:v) saline and glycerol to a final volume of 1.7 mLjust prior to activation. The air headspace was then exchanged with PFP,sealed with a West grey butyl stopper, the vial crimped with an aluminumseal, activated and tested for microsphere number and average size asdescribed in Example 7. ^(c)Vials were diluted with saline to a finalvolume of 1.7 mL just prior to activation. The air headspace was thenexchanged with PFP, sealed with a West grey butyl stopper, the vialcrimped with an aluminum seal, activated and tested for microspherenumber and average size as described in Example 7. ^(d)Vials (2 ccWheaton vials) were prepared by diluting DEFINITY ® with formulationmatrix 1 to 4 or 1 to 2 (undiluted DEFINITY ® was also tested), theheadspace gas exchanged with PFP, the vials stoppered and crimped withan aluminum seal, activated and tested for microsphere number andaverage size as described in Example 7.

TABLE 17 Microsphere characteristics with different Lipid mg/mL PG/Gformulations^(a) Micro- Lipid Blend Lipid Blend sphere concentrationTime of concentration per mL Diameter in formulation Dilution afterdilution, (×10⁹) (μm) DEFINITY ® n/a n/a 3.05 1.66 0.75 mg Lipid Blendper mL^(d) 3.75 mg Lipid Before  0.75 mg/mL 4.71 1.66 Blend/mL PG/GActivation^(b) 3.75 mg Lipid After  0.75 mg/mL 3.12 1.60 Blend/mL PG/GActivation^(c) DEFINITY ® Before  0.375 mg/mL 1.38 1.66 diluted toactivation 0.375 mg Lipid Blend per mL^(d) 1.875 mg Lipid Before  0.375mg/mL 2.45 1.74 Blend/mL PG/G Activation^(b) 1.875 mg Lipid After  0.375mg/mL 1.73 1.66 Blend/mL PG/G Activation^(c) DEFINITY ® before 0.1875mg/mL 0.54 1.75 diluted to activation 0.1875 mg Lipid Blend per mL^(d)0.9375 mg Lipid Before 0.1875 mg/mL 1.00 1.72 Blend/mL PG/GActivation^(b) 0.9375 mg Lipid After 0.1875 mg/mL 0.41 1.89 Blend/mLPG/G Activation^(c) ^(a)Vials (2 cc Wheaton vials) were prepared byweighting 391 mg of 1:1 (v/v) propylene glycol and glycerol containing0.9375, 1.875 or 3.75 mg lipid blend/mL (ratios of 1:552:672; 1:276:336;and 1:138:168 for LB:PG:G, respectively). ^(b)Vials were diluted withsaline to a final volume of 1.7 mL just prior to activation. The airheadspace was then exchanged with PFP, sealed with a West grey butylstopper, the vial crimped with an aluminum seal, activated and testedfor microsphere number and average size as described in Example 7.^(c)The air headspace was exchanged with PFP, the vial sealed with aWest grey butyl stopper, crimped with an aluminum seal and activated.Saline was added to a final volume of 1.7 mL and the vial tested formicrosphere number and average size as described in Example 7. ^(d)Vials(2 cc Wheaton vials) were prepared by diluting DEFINITY ® withformulation matrix 1 to 4 or 1 to 2 (undiluted DEFINITY ® was alsotested), the headspace gas exchanged with PFP, the vials stoppered andcrimped with an aluminum seal, activated and tested for microspherenumber and average size as described in Example 7.

These studies demonstrated that lipid blend formulations havingdifferent lipid concentrations, when activated, produce proportionalnumbers of microspheres (per given volume, 1 mL). The microsphere sizewas equivalent to activated DEFINITY® and microsphere number similar orhigher than activated DEFINITY® or an equivalent diluted form. Theability to form microspheres with characteristics equivalent toactivated DEFINITY® with a variety of different lipid blendconcentrations in PG or PG/G was not expected. The ability to achievethis by activating lipid blend in PG/G before the addition of diluentswas even further surprising.

Example 11. Containers

New lipid formulations or DEFINITY® were filled into various containersincluding: vials, syringes, and pliable plastic tubes, which were thenactivated. In all studies, an appropriate amount of lipid formulationwas placed in the container, the headspace replaced with PFP, thecontainer sealed, and the formulation activated.

TABLE 18 Microsphere characteristics for lipid blend in PG or PG/Gformulations activated in 2 mL Schott vial^(a) Volume of Micro- SalineMicrosphere sphere Time of Dilution Mean Diameter per mL Fill WeightDilution (mL) (microns) (×10⁹) 55 mg of 7.5 mg Before 0.50 1.52 6.23LB/mL of PG Activation 89 mg of 7.5 mg Before 0.80 1.52 4.83 LB/mL of PGActivation 134 mg of 7.5 mg Before 1.20 1.61 5.29 LB/mL of PG Activation177 mg of 7.5 mg Before 1.59 1.63 5.00 LB/mL of PG Activation 122 mg of3.75 mg Before 0.43 1.57 5.43 LB/mL of PG/G Activation 196 mg of 3.75 mgBefore 0.69 1.55 5.31 LB/mL of PG/G Activation 295 mg of 3.75 mg Before1.04 1.61 4.48 LB/mL of PG/G Activation 392 mg of 3.75 mg Before 1.381.60 4.96 LB/mL of PG/G Activation 196 mg of 3.75 mg After 0.69 1.881.77 LB/mL of PG/G Activation 295 mg of 3.75 mg After 1.04 1.69 2.68LB/mL of PG/G Activation 392 mg of 3.75 mg After 1.38 1.56 4.06 LB/mL ofPG/G Activation ^(a)The appropriate amount of 7.5 mg LB/mL PG or 3.75 mgLB per mL PG/G formulation was weighed into a 2 mL Schott vial, anappropriate amount of saline added for “before activation” samples, theair headspace replaced with PFP, the vial sealed with West grey butylstoppers, crimped with an aluminum seal, activated, an appropriateamount of saline added for “after activation samples”, and tested formicrosphere number and average size as described in Example 7. Vialswere activated using a VIALMIX ®.

TABLE 19 Microsphere characteristics for lipid blend in PG or PG/Gformulations activated in 1 mL Wheaton V-vial^(a) Volume of Micro-Saline Microsphere sphere Time of Dilution Mean Diameter per mL FillWeight Dilution (mL) (microns) (×10⁹) 55 mg of 7.5 mg Before 0.50 1.646.33 LB/mL of PG Activation 88 mg of 7.5 mg Before 0.80 1.73 4.04 LB/mLof PG Activation 177 mg of 7.5 mg Before 1.59 1.63 5.00 LB/mL of PGActivation 122 mg of 3.75 mg Before 0.43 1.57 5.28 LB/mL of PG/GActivation 392 mg of 3.75 mg Before 1.38 1.60 4.96 LB/mL of PG/GActivation 122 mg of 3.75 mg After 0.43 1.78 1.06 LB/mL of PG/GActivation 392 mg of 3.75 mg After 1.38 1.68 3.07 LB/mL of PG/GActivation ^(a)The appropriate amount of 7.5 mg LB/mL PG or 3.75 mgLB/mL PG/G formulation was weighed into a 1 mL Wheaton V-vial ,the airheadspace replaced with PFP, an appropriate amount of saline added for“before activation” samples, the vial sealed with West grey butylstoppers, crimped with an aluminum seal, activated, an appropriateamount of saline added for “after activation” samples, and tested formicrosphere number and average size as described in Example 7. Vialswere activated using a VIALMIX ®.

TABLE 20 Microsphere concentration for DEFINITY ® activated insyringes^(a) Volume Microsphere Microsphere Syringe (mL) of MeanDiameter per mL Size DEFINITY ® (microns) (×10⁹) 3 mL 1.5 1.63 2.45 5 mL1.6 1.78 0.961 5 mL 1.9 1.92 1.00 5 mL 2.25 1.76 2.25 5 mL 2.7 1.780.513 ^(a)DEFINITY ® filled (1.5 to 2.7 mL) into 3 and 5 mL NORM-JECT ®syringes ((Henke-Sass, Wolf GmbH, Tuttlingen, Germany)), The airheadspace was replaced with PFP, the syringe activated with aWig-L-Bug ™, tested for microsphere number and average size as describedin Example 7.

TABLE 21 Microsphere characteristics for lipid blend in PG or PG/Gformulations activated in 3 mL NORM-JECT ® syringe Volume of Micro-Saline Microsphere sphere Time of Dilution Mean Diameter per mL FillWeight Dilution (mL) (microns) (×10⁹) 101 mg of 7.5 mg Before 0.90 1.794.02 LB/mL of PG Activation 177 mg of 7.5 mg Before 1.59 1.66 4.15 LB/mLof PG Activation 222 mg of 7.5 mg Before 0.78 1.72 3.63 LB/mL of PGActivation 222 mg of 3.75 mg After 0.78 1.57 4.83 LB/mL of PG/GActivation ^(a) The appropriate amount of 7.5 mg LB/mL PG or 3.75 mgLB/mL PG/G formulation was weighed into a 3 mL NORM-JECT ® syringe((Henke-Sass, Wolf GmbH, Tuttlingen, Germany), an appropriate amount ofsaline added for “before activation” samples, the air headspace replacedwith PFP and the syringe activated, an appropriate amount of salineadded for “after activation” samples, and the preparations tested formicrosphere number and average size as described in Example 7. Syringeswere activated using a VIALMIX ®.

TABLE 22 Microsphere characteristics for lipid blend in PG Formulationsactivated in syringe modified to have two compartments formed with adental amalgam capsule^(a) Volume of Micro- Saline Microsphere sphereTime of Dilution Mean Diameter per mL Fill Weight^(b) Dilution (mL)(microns) (×10⁹) 177 mg of 7.5 mg Before 1.59 1.66 4.15 LB/mL of PGActivation 391 mg of 3.75 mg Before 1.38 1.64 4.14 LB/mL of PG/GActivation 391 mg of 3.75 mg After 1.38 1.59 3.92 LB/mL of PG/GActivation ^(a)A 5 mL NORM-JECT ® syringe (Henke-Sass, Wolf GmbH,Tuttlingen, Germany) was cut down to 3 mL. A dental amalgam capsule(obtained from a local dentist) was opened, the bottom containing powderwas removed, and the plunger was removed from the top compartment alongwith the contents of the top compartment. The top compartment was fittedinto the barrel of the cut down syringe. ^(b)The appropriate amount of7.5 mg LB/mL PG or 3.75 mg LB/mL PG/G formulation was weighed into thebody of a 5 mL NORM-JECT ® syringe cut down to approximately 2.5 mL((Henke-Sass, Wolf GmbH, Tuttlingen, Germany), the dental amalgamplunger was inserted into the capsule, an appropriate amount of salineadded for “before activation” samples, the air headspace replaced withPFP, the syringe sealed with a luer lock cap, activated, an appropriateamount of saline added for “after activation” samples, and tested formicrosphere number and average size as described in Example 7. Vialswere activated using a VIALMIX ®.

TABLE 23 Microsphere characteristics for lipid blend in PG or PG/Gformulations activated in syringe modified to have two compartments^(a)Volume of Micro- Saline Microsphere sphere Time of Dilution MeanDiameter per mL Fill Weight^(b) Dilution (mL) (microns) (×10⁹) 60 mg of7.5 mg Before 0.54 1.83 4.74 LB/mL of PG Activation 133 mg of 3.75 mgBefore 0.47 1.72 4.42 LB/mL of PG/G Activation 133 mg of 3.75 mg After0.47 1.89 1.40 LB/mL of PG/G Activation ^(a)A 3 mL NORM-JECT ® syringe(Henke-Sass, Wolf GmbH, Tuttlingen, Germany) was modified to have anapproximate 3 mm by 10 mm × 1 mm bulge as a bypass channel typical ofcommercial two compartment syringes. The channel was made by heating onetong of a forceps and pressing it to the inside of the syringe barrel atabout the 2 mL volume mark. The end of a syringe plunger was cut off toa length of approximately 1 cm and used as the bypass plug. A secondsyringe plunger was also cut down. ^(b)The appropriate amount of 7.5 mgLB/mL PG or 3.75 mg LB/mL PG/G formulation was weighed into the body ofthe modified 3 mL NORM-JECT ® syringe below the bypass channel, thebypass plug was inserted to a point just above the bypass channel, anappropriate amount of saline was added to the top chamber formed afterinsertion of the bypass plug, the cutdown syringe plunger was insertedcompleting the fill of the upper chamber. The air headspace in the lowerchamber was replaced with PFP, the syringe sealed with a luer lock cap.The syringe was activated using a VIALMIX ®, the syringe plunger pushedto move the bypass plug to the bypass channel, allowing the saline toenter the lower chamber containing activated product for the “afteractivation” samples. For the “before activation” samples, the syringeplunger was pushed to move the bypass plug to the bypass channel,allowing the saline to enter the lower chamber the syringe activatedusing a VIALMIX ®. Samples were tested for microsphere number andaverage size as described in Example 7.

TABLE 24 Microsphere characteristics for lipid blend PG or PG/Gformulations activated in two compartment plastic tube Volume of Micro-Saline Microsphere sphere Time of Dilution Mean Diameter per mL FillWeight^(a) Dilution (mL) (microns) (×10⁹) 177 mg of 7.5 mg Before 1.591.64 3.11 LB/mL of PG Activation 392 mg of 3.75 mg Before 1.38 1.80 2.62LB/mL of PG/G Activation 392 mg of 3.75 mg After 1.38 1.63 3.72 LB/mL ofPG/G Activation ^(a)The appropriate amount of 7.5 mg LB/mL PG or 3.75 mgLB/mL PG 7 G formulation was weighed into the lower chamber of a twocompartment tube (NEOPAC Fleximed Tube, 13.5 × 80 mm, Hoffmann NeopacAG, Oberdiessbach, Switzerland), an appropriate amount of saline wasadded to the top chambert, the air headspace in the lower chamber wasreplaced with PFP, the tube sealed with a luer lock cap. The tube wasactivated using a VIALMIX ®, the upper compartmet saline was transferredto the lower compartment and mixed, for the “after activation” samples.For the ‘before activation” samples, the saline was tranferred to thelower compartment before the tube activation using a VIALMIX ®. Sampleswere tested for microsphere number and average size as described inExample 7.

These studies demonstrated that the process of shaking lipid blend PGand PG/G formulations with a mechanical shaker could be achieved in avariety of containers including vials, syringes and a plastic tube andproduce microspheres with characteristics equivalent to activatedDEFINITY®. Surprisingly, the mechanical shaking overcame differences inthe dimensions of the container and the material the container was madefrom and allowed the formation of microspheres with equivalent size andnumber to be formed. Activation of the LB formulations in PG and PG/G insyringes both before and after addition of diluents is an excitingfinding. In addition being able to separate the diluents from theformulation and then allow the two components to come together prior toor after activation by mechanical shaking proves new opportunities toprovide preparations that can achieve a room temperature stableformulation with an easy product production.

Example 12. Activation Methods

Studies were conducted to demonstrate the ability to activate DEFINITY®with several methods other than use of the VIALMIX®. These methods aredescribed below with results reported in Table 25.

-   -   A. DEFINITY® (1.5 mL) was drawn into a 3 ml plastic syringe and        connected to a 3 way stopcock. A separate syringe of the same        size was filled with PFP gas and connected to another port on        the stopcock. The DEFINITY® and PFP gas were mixed by        alternately depressing the plunger on each syringe back and        forth between 50-400 times. Microbubble count and bubble        diameter measurements indicate activation of DEFINITY®.    -   B. DEFINITY® (3.0 mL) was drawn into a 10 ml plastic syringe and        connected to a 3 way stopcock. A separate syringe of the same        size was filled with PFP gas and connected to another port on        the stopcock. The DEFINITY® and PFP gas were mixed by        alternately depressing the plunger on each syringe back and        forth 200 times. Microbubble count and bubble diameter        measurements indicate activation of DEFINITY®.    -   C. A lipid formulation (1.5 mL of 0.045 mg/mL DPPA, 0.75 mg/mL        DPPC, 0 mg/mL MPEG5000DPPE, 4.87 mg NaCl/mL, 103.5 mg/mL        propylene glycol, 126.2 mg/mL glycerol, 2.34 mg/mL NaH₂PO₄H₂O,        2.16 mg/mL NaHPO₄7H₂O) was drawn into a 3 ml plastic syringe and        connected to a 3 way stopcock. A separate syringe of the same        size was filled with PFP gas and connected to another port on        the stopcock. The formulation and PFP gas were mixed by        alternately depressing the plunger on each syringe back and        forth 100 times. Microbubble count and bubble diameter        measurements indicate activation of this lipid formulation.    -   D. A modified lipid formulation (1.5 mL of 0.045 mg/mL DPPA,        0.75 mg/mL DPPC, 0 mg/mL MPEG5000DPPE, 4.87 mg/mL NaCl, 103.5        mg/mL propylene glycol, 126.2 mg/mL glycerol, 2.34 mg/mL        NaH₂PO₄H₂O, 2.16 mg/mL NaHPO₄7H₂O) was drawn into a 3 ml plastic        syringe and connected to a 3 way stopcock. A separate syringe of        the same size was filled with PFP gas and connected to another        port on the stopcock. In between the lipid formulation filled        syringe and the stopcock was a plastic tube filed with seven        high performance X-grid static mixers (StaMixCo, GXP-9, 4-PA66,        black). The formulation and PFP gas were mixed by alternately        depressing the plunger on each syringe back and forth 50 times.        Microbubble count and bubble diameter measurements indicate        activation of this lipid formulation.    -   E. A modified lipid formulation (1.5 mL of 0.045 mg/mL DPPA,        0.75 mg/mL DPPC, 0 mg/mL MPEG5000DPPE, 4.87 mg/mL NaCl, 103.5        mg/mL propylene glycol, 126.2 mg/mL glycerol, 2.34 mg/mL        NaH₂PO₄H₂O, 2.16 mg/mL NaHPO₄7H₂O) was drawn into a 3 ml plastic        syringe and connected to two 3 way stopcocks in series. A        separate syringe of the same size was filled with PFP gas and        connected to another port on the stopcock. The formulation and        PFP gas were mixed by alternately depressing the plunger on each        syringe back and forth 100 times. Microbubble count and bubble        diameter measurements indicate activation of this lipid        formulation.    -   F. A modified lipid formulation (1.5 mL of 0.045 mg/mL DPPA,        0.75 mg/mL DPPC, 0 mg/mL MPEG5000DPPE, 4.87 mg/mL NaCl, 103.5        mg/mL propylene glycol, 126.2 mg/mL glycerol, 2.34 mg/mL        NaH₂PO₄H₂O, 2.16 mg/mL NaHPO₄7H₂O) was drawn into a 3 ml plastic        syringe and connected to a 3 way stopcock. A separate syringe of        the same size was filled with PFP gas and connected to another        port on the stopcock. In between the lipid formulation filled        syringe and the stopcock was a plastic tube filed with eight        high performance X-grid static mixers (StaMixCo, GXF-10-2-ME,        orange). The formulation and PFP gas were mixed by alternately        depressing the plunger on each syringe back and forth 100 times.        Microbubble count and bubble diameter measurements indicate        activation of this lipid formulation.    -   G. A modified lipid formulation (1.5 mL of 0.045 mg/mL DPPA,        0.401 mg/mL DPPC, 0.304 mg/mL MPEG5000DPPE, 4.87 mg/mL NaCl,        155.25 mg/mL propylene glycol, 31.55 mg/mL glycerol, 2.34 mg/mL        NaH₂PO₄H₂O, 2.16 mg/mL NaHPO₄7H₂O) was drawn into a 3 ml plastic        syringe and connected to a 3 way stopcock. A separate syringe of        the same size was filled with PFP gas and connected to another        port on the stopcock. The formulation and PFP gas were mixed by        alternately depressing the plunger on each syringe back and        forth 100 times. Microbubble count and bubble diameter        measurements indicate activation of this lipid formulation.    -   H. DEFINITY® (1.5 mL) plus 3.5 mL of saline were drawn into a 5        ml plastic syringe and connected to a 3 way stopcock. A separate        syringe of the same size was filled with PFP gas and connected        to another port on the stopcock. The DEFINITY®, saline and PFP        gas were mixed by alternately depressing the plunger on each        syringe back and forth 100 times. Microbubble count and bubble        diameter measurements indicate activation of DEFINITY®.    -   I. DEFINITY® (1.5 mL) was drawn into a 3 ml plastic syringe and        connected to a 3 way stopcock. A separate syringe of the same        size was filled with PFP gas and connected to another port on        the stopcock. In between the DEFINITY® filled syringe and the        stopcock was a plastic tube with a plastic helical mixer        (StaMixCo, 2.5″× 3/16″, 15 helical turns in 2.5 inches). The        DEFINITY® and PFP gas were mixed by alternately depressing the        plunger on each syringe back and forth 50 times. Microbubble        count and bubble diameter measurements indicate activation of        DEFINITY®.    -   J. DEFINITY® (3.0 mL) was drawn into a 10 ml plastic syringe and        connected to a 3 way stopcock. A separate syringe of the same        size was filled with PFP gas and connected to another port on        the stopcock. In between the DEFINITY® filled syringe and the        stopcock was a plastic tube with a plastic helical mixer        (StaMixCo, 2.5″× 3/16″, 15 helical turns in 2.5 inches). The        DEFINITY® and PFP gas were mixed by alternately depressing the        plunger on each syringe back and forth 50 times. Microbubble        count and bubble diameter measurements indicate activation of        DEFINITY®.    -   K. DEFINITY® (1.5 mL) was drawn into a 3 ml plastic syringe and        connected directly to a plastic tube with a plastic helical        mixer (StaMixCo, 2.5″× 3/16″, 15 helical turns in 2.5 inches). A        separate syringe of the same size was filled with PFP gas and        connected to the other end of the plastic tube. The DEFINITY®        and PFP gas were mixed by alternately depressing the plunger on        each syringe back and forth 25 times. Microbubble count and        bubble diameter measurements indicate activation of DEFINITY®.    -   L. DEFINITY® (1.5 mL) was drawn into a 3 ml plastic syringe and        connected directly to a 20 u QMA filter (Waters). A separate        syringe of the same size was filled with PFP gas and connected        to the other end of the filter. The DEFINITY® and PFP gas were        mixed by alternately depressing the plunger on each syringe back        and forth 50 times. Microbubble count and bubble diameter        measurements indicate activation of DEFINITY®.    -   M. DEFINITY® (1.5 mL) was drawn into a 3 ml plastic syringe and        connected directly to a 20 u QMA filter (Waters) and plastic        tube with a plastic helical mixer (StaMixCo, 2.5″× 3/16″, 15        helical turns in 2.5 inches). A separate syringe of the same        size was filled with PFP gas and connected to the other end of        the mixer. The DEFINITY® and PFP gas were mixed by alternately        depressing the plunger on each syringe back and forth 50 times.        Microbubble count and bubble diameter measurements indicate        activation of DEFINITY®.    -   N. DEFINITY® (0.6 mL) was drawn into a 1 ml glass syringe and        connected to a 1.5 inch metal holder containing a 5 u filter. A        separate glass syringe of the same size was filled with PFP gas        and connected to the other end of the metal holder. This        extrusion device is commercial available (LiposoFast-Basic,        Avestin, Inc.) The DEFINITY® and PFP gas were mixed by        alternately depressing the plunger on each syringe back and        forth 25 times. Microbubble count and bubble diameter        measurements indicate activation of DEFINITY®.    -   O. DEFINITY® (0.6 mL) was drawn into a 1 ml glass syringe and        connected to a 1.5 inch metal holder containing a 5 u filter. A        separate glass syringe of the same size was filled with PFP gas        and connected to the other end of the metal holder. This        extrusion device is commercial available (LiposoFast-Basic,        Avestin, Inc.) The DEFINITY® and PFP gas were mixed by        alternately depressing the plunger on each syringe back and        forth 100 times. Microbubble count and bubble diameter        measurements indicate activation of DEFINITY®.    -   P. DEFINITY® (0.6 mL) was drawn into a 1 ml glass syringe and        connected to a 1.5 inch metal holder containing either a 0.4 or        1.0 micron filter. A separate glass syringe of the same size was        filled with PFP gas and connected to the other end of the metal        holder. This extrusion device is commercial available        (LiposoFast-Basic, Avestin, Inc.) The DEFINITY® and PFP gas were        mixed by alternately depressing the plunger on each syringe back        and forth 25 times. Microbubble count and bubble diameter        measurements indicate activation of DEFINITY®.    -   Q. A vial of DEFINITY® (1.5 mL) was vortexed at the highest        setting for 5 minutes. Microbubble count and bubble diameter        measurements indicate activation of DEFINITY®.    -   R. A vial of DEFINITY® (1.5 mL) was sonicated for 2 minutes. The        solution was a milky white, however was not tested for        microbubble count or bubble diameter measurements.    -   S. A vial of DEFINITY® (1.5 mL) was treated for 5 minutes with a        high speed blade homogenizer. The solution was a milky white,        however was not tested for microbubble count or bubble diameter        measurements.    -   T. A vial of DEFINITY® (1.5 mL) was secured on the end of a        0.75″×2.25″×23″ wood stick, moved between two wood posts 15″        apart between 300 and 1500 times at rate of 100 hits/27 seconds,        and tested for microbubble count or bubble diameter        measurements. Microbubble count and bubble diameter measurements        indicate activation of DEFINITY®.

TABLE 25 Results of microbubble counts and diameter using a Sysmexmicrobubble analyzer. # of syringe barrel depressions back and forth #Microbubbles Microbubble (Example “T” (×10⁹) Diameter Example is # hits)per ml (microns) A  50 0.80, 1.49 1.8-2.0  75 1.19 1.7 100 0.57, 1.25,1.40 1.8, 1.9 200 1.28 1.7 400 1.02 1.6 B 200 0.55 1.7 C 100 1.28 1.7 D 50 0.50 1.9 E 100 0.99 1.7 F 100 0.69 2.0 G 100 1.08 2.0 H 100 0.08 2.3I  50 0.23 1.9 J  50 0.16 1.9 K  25 0.14 2.0 L  50 0.07 2.1 M  50 0.112.1 N  25 0.10 1.7 O 100 0.57 1.3 P 0.4 u and 1.0 u 0.01 and 0.12 3.6and 1.9 filter 25 Q Vortex 5 min 0.13 2.1 R Sonicate 2 min Not testedNot tested based on visual- based on visual- Light Milky Light Milky SPolytron 5 min Not tested Not tested based on visual- based on visual-Light Milky Light Milky T Between 300 and 1500 times at rate of 100hits/27 seconds  300x  0.056  1.86  500x  0.096  1.93 1000x  0.205  1.901500x  0.194  1.74

These studies demonstrate activation of DEFINITY® or modified versionsthereof can be accomplished using a variety of activation devices.

The references recited herein, including patents and patentapplications, are incorporated by reference in their entirety.

What is claimed is:
 1. A composition of an ultrasound contrast agentcomprising a non-aqueous mixture comprising DPPA, DPPC and MPEG5000-DPPEin propylene glycol and/or glycerol, and a perfluorocarbon gas, whereinthe non-aqueous mixture comprises less than 5% w/w (weight/weight)water, and wherein DPPA, DPPC and MPEG5000-DPPE are present in a ratioof about 10:82:8 (mole %).
 2. The composition of claim 1, wherein thecomposition comprises less than 10% impurities relative to total lipidcontent when stored at room temperature for about 3 months.
 3. Thecomposition of claim 1, wherein the composition comprises less than 5%impurities relative to total lipid content when stored at roomtemperature for about 3 months.
 4. The composition of claim 1, whereinthe composition comprises less than 2% impurities relative to totallipid content when stored at room temperature for about 3 months.
 5. Thecomposition of claim 2, wherein the impurities are measured using HPLC.6. The composition of claim 1, wherein the non-aqueous mixture comprisesDPPA, DPPC and MPEG5000-DPPE in propylene glycol.
 7. The composition ofclaim 6, wherein the weight ratio of DPPA, DPPC and MPEG5000-DPPEcombined to propylene glycol is in a range of about 1:100 to about1:600.
 8. The composition of claim 1, wherein the non-aqueous mixturecomprises DPPA, DPPC and MPEG5000-DPPE in propylene glycol and glycerol.9. The composition of claim 8, wherein the weight ratio of DPPA, DPPCand MPEG5000-DPPE combined to propylene glycol to glycerol is in a rangeof about 1:100:100 to about 1:600:700.
 10. The composition of claim 1,wherein the non-aqueous mixture comprises DPPA, DPPC and MPEG5000-DPPEin glycerol.
 11. The composition of claim 10, wherein the weight ratioof DPPA, DPPC and MPEG5000-DPPE combined to glycerol is about 1:100 toabout 1:700.
 12. The composition of claim 1, wherein the compositionfurther comprises a buffer.
 13. The composition of claim 1, wherein thecomposition further comprises a non-phosphate buffer.
 14. Thecomposition of claim 1, wherein DPPA, DPPC and MPEG5000-DPPE combinedare present in a concentration of about 0.1 to about 10 mg per ml ofnon-aqueous mixture.
 15. The composition of claim 1, wherein theperfluorocarbon gas is perfluoropropane gas.
 16. The composition ofclaim 1, wherein the composition consists essentially of DPPA, DPPC,MPEG5000-DPPE, propylene glycol, and perfluorocarbon gas.
 17. Thecomposition of claim 1, wherein the composition is provided in a vial.18. The composition of claim 1, wherein the composition is provided in avial with a V-bottom.
 19. The composition of claim 1, wherein thecomposition is provided in a vial with a flat-bottom.
 20. Thecomposition of claim 1, wherein the composition is provided in a vialwith a rounded-bottom.
 21. The composition of claim 1, wherein thecomposition is provided in a single chamber container.
 22. Thecomposition of claim 1, wherein the composition is provided in amultiple chamber container.
 23. A container comprising the compositionof claim 1 in a first chamber and an aqueous diluent in a secondchamber.
 24. The container of claim 23, wherein the aqueous diluent isan aqueous buffered saline solution.